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Anthropogenic and naturally produced brominated phenols in pet blood and pet food in Japan Hazuki Mizukawa, Kei Nomiyama, Susumu Nakatsu, Miyuki Yamamoto, Mayumi Ishizuka, Yoshinori Ikenaka, Shouta MM Nakayama, and Shinsuke Tanabe Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01009 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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Environmental Science & Technology

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Research article

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Anthropogenic and naturally produced brominated phenols in pet blood and pet food in Japan

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Hazuki Mizukawa†,1, Kei Nomiyama†,*, Susumu Nakatsu‡, Miyuki Yamamoto†, Mayumi Ishizuka§, Yoshinori Ikenaka§,ǁ, Shouta M.M. Nakayama§ and Shinsuke Tanabe†

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†

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama,

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Ehime 790-8577, Japan

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‡

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Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan

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Nakatsu Veterinary Surgery, 2-2-5, Shorinjichonishi, Sakai-ku, Sakai-shi, Osaka 590-0960, Japan

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Water Research Group, Unit for Environmental Sciences and Management, North-West University, 53 Borcherd Street, Potchefstroom 2531, South Africa

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*Address correspondence to:

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Kei Nomiyama,

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Tel/Fax : +81-89-927-8171

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E-mail: [email protected]

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Present address: Department of Environmental Veterinary Science, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan

ACS Paragon Plus Environment


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Abstract

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Present study determined concentrations and residue patterns of bromophenols (BPhs) in whole

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blood samples of pet cats and pet dogs collected from veterinary hospitals in Japan. BPhs

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concentrations were higher in cat blood than in dog blood, with statistically insignificant differences

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(p = 0.07). Among the congeners, 2,4,6-tribromophenol (TBPh) constituted the majority of BPhs

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(>90%) detected in both species. Analysis of commercial pet food to estimate exposure routes

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showed that the most abundant congener in all pet food samples was 2,4,6-TBPh, accounting for

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>99% of total BPhs. This profile is quite similar to the blood samples of the pets, suggesting that diet

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might be an important exposure route for BPhs in pets. After incubation in polybrominated diphenyl

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ether (PBDE) mixtures (BDE-47, BDE-99 and BDE-209), 2,4,5-TBPh was found in dog liver

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microsomes but not in cat liver microsomes, implying species-specific metabolic capacities for

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PBDEs. Formation of 2,4,5-TBPh occurred by hydroxylation at the 1′ carbon atom of the ether bond

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of BDE-99 is similar with human study reported previously. Hydroxylated PBDEs were not detected

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in cats or dogs; therefore, diphenyl ether bond cleavage of PBDEs can also be an important

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metabolic pathway for BPhs formation in cats and dogs.

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TOC 40



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Introduction

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Brominated phenols (BPhs) that have simple aromatic structures are discharged into the

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environment via anthropogenic and natural sources.1 2,4,6-tribromophenol (TBPh) is used as a

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reactive flame retardant intermediate and as a wood preservative.1-2 Pentabromophenol (PBPh) is

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used as a molluscicide and an intermediate in the production of pentabromophenoxy compounds.1-2

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BPhs are also produced naturally in the marine environment. In particular, mono-, di-, and tri-BPhs

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are excreted by marine organisms such as algae, cyanobacteria, sponges, and polychaetes3, and

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several species of marine algae are known to contain and biosynthesize BPhs.4-5 The congeners

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4-monobromophenol (MBPh), 2,4-dibromophenol (DBPh), 2,6-DBPh, and 2,4,6-TBPh have been

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reported as naturally occurring BPhs.4, 6-7 Moreover, BPhs can be formed from the biotransformation

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of other brominated contaminants, such as brominated benzenes and polybrominated diphenyl ethers

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(PBDEs) catalyzed by cytochrome P450 (CYP) enzymes.3, 8 2,4-DBPh and 2,4,5-TBPh were found

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as metabolites in human liver tissues after in vitro exposure to BDE-47 and BDE-99, respectively.9-11

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2,4,5-TBPh was found to be one of the major metabolites of BDE-99 in human liver microsomes.

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The formation of hydroxylated metabolites was catalyzed solely by CYP2B6.11 In the case of rats,

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formation of 2,4,5-TBPh from BDE-99 was catalyzed by eight types of CYP enzymes including

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CYP2A2, 2C12, and 3As.12 Previous studies reported that 2,4,6-TBPh and PBPh were very potent

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T4 competitors in the binding assay that use human transthyretin (TTR) with 10 and 7.1 times higher

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TTR-binding potency than the natural ligand T4, respectively.13-14 Therefore, BPhs may disrupt


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thyroid hormone (TH) homeostasis. A very recent study reported that mice treated with 2,4,6-TBPh

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showed decreased levels of deiodinase 1 and TH receptor β isoform 2 and increased levels of

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deiodinase 2 and growth hormone mRNA.15 Moreover, the relative mRNA levels of thyroid-

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stimulating hormone β increased in the pituitary gland.15

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Domestic pets such as dogs and cats share living environments with humans. Therefore, they are

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exposed to various contaminants, including brominated flame retardants (BFRs), in their immediate

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surroundings, which raise concerns about risk to their health. In fact, high levels of polybrominated

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diphenyl ethers (PBDEs), a typical BFR found in the blood of cats in Japan, USA, Sweden, UK,

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Pakistan, and Australia, were reported in recent years, and the highest levels of PBDEs were

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detected in the blood of American cats.16-21 The residual levels of blood PBDE levels in the Japanese

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cats (180 pg/g wet wt.) are 1-3 orders of magnitude lower than those reported for the serum of pet

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cats in the USA (3.5~6.2 ng/ml in serum).19, 22 Likewise, while dogs generally have lower levels than

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cats,17 PBDE concentrations in dogs from Japan (100 pg/g wet wt.) are approximately one-eighth

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that of US pet dogs (860 pg/g wet wt.).19, 23 2,4-DBPh, 2,4,6-TBPh, and PBPh have been identified

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as flame retardants;24 therefore, these compounds are commonly found in the indoor environment

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where humans and pet animals spend long hours. In fact, indoor air collected from Japanese modern

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houses contained larger amounts of 2,4,6-TBPh than higher-brominated PBDEs (penta-BDEs to

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deca-BDE) and hexabromocyclododecanes (HBCDs).25 Therefore, special attention should be given

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to the exposure to 2,4,6-TBPh via indoor air inhalation and dust ingestion.25



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Significant routes of exposure to BPhs for pet dogs and cats are through pet food and house dust.

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For pet food exposure route, it is considered that pet dogs and cats are ingested BPhs through the pet

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food, in which fish meat, containing naturally occurring BPhs, is the primary ingredient. In our

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previous study, methoxylated PBDEs (MeO-PBDEs), also identified as natural compounds in the

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marine environment, were detected in the blood of cats and cat food,19 suggesting that natural origin

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BPhs may be detected from the pet food contained sea-food materials. Total PBDEs, in the USA, a

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high mean concentration of 0.17-1.75 ng g-1 wet wt. in canned cat food and 0.6-2.9 ng g-1 wet wt. in

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dry cat food were found.22 Compare to USA, lower levels of total PBDEs in cat food were found in

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France (0.088 ng g-1 wet wt. in pellet) and Japan (0.21 ng g-1 wet wt. in dry food and 0.71 ng g-1 wet

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wt. in wet food).19, 26 There are also found the country difference of cat food taste (e.g. seafood

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dominant in Japan; meat and seafood are comprising in USA) and/or accumulation pattern,

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suggesting that regional specific exposure pattern will be existed. For house dust exposure route, pet

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dogs and cats are ingested contaminated house dust, and they are expected to have higher exposure

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to BPhs because of increased intake of house dust from their grooming behavior and/or inhalation.

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Additional important route as the metabolism of BFRs such as PBDEs will also be suspected.

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However, there have been only two prior studies that analyzed BPhs in the blood of pet dogs and

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cats,17, 20 and no information is available on the levels of BPhs in pet food to estimate their exposure

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route and biotransformation ability from PBDEs to BPhs in dogs and cats. Other previous report also


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indicated that contaminant intake by food ingestion was approximately 100-fold higher than that by

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indoor air inhalation.26

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This study investigates the levels and accumulation patterns of BPhs in the blood samples of pet

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dogs and cats collected from a veterinary hospital in Japan. To estimate the routes of exposure to

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these chemicals, we analyzed the representative samples (popular pet food brands and purchasable

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everywhere in Japan), of dry and wet pet food products to identify dietary exposure pathways. In

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addition, we conducted in vitro metabolic assays to confirm the biotransformation of PBDEs to

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BPhs by the CYPs in the livers of dogs and cats. Finally, we compared the biotransformation

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capacity of BPhs in these pet animals.

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Materials and methods

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Sample Collection

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The details of pet blood samples (dogs: n = 17, cats: n = 11) and pet food samples (dry food: n = 8,

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wet food: n = 8) are described in Table S1, S2 and Mizukawa et al. (2016).19 Briefly, whole blood

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samples were collected at the Nakatsu Veterinary Surgery in Osaka and the Tao Veterinary Hospital

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in Hiroshima, Japan, during 2009-2012. In this study, blood collection were performed by a

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Veterinarian during a routine consultation, so we couldn't collect it except for whole blood to prevent

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the medical treatment interruption. The pets were brought to the veterinary hospitals for clinical

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treatments such as surgical procedures (for lymphoma and pyometra, neutering etc.), but excluding



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feline hyperthyroidism (FH). The pet owners completed a questionnaire, providing information about

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the pet’ age, sex, weight, housing conditions, eating habits (dry or wet food), and housing

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environment (indoors or outdoors). Commercial dry and wet pet food products were purchased from

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Japanese pet shops in 2010.19

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The pooled liver microsome from ten healthy beagle dogs used for the in vitro metabolic assay was

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purchased from Life Technologies (Carlsbad, CA, USA). We collected fresh liver samples (within 30

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min of the donor’s death) from three domestic cats, with the cooperation of Nakatsu Veterinary

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Surgery in Osaka, Japan.19 These cats were euthanized using pentobarbital sodium because of

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incurable (and painful) diseases. Owners provided consent for harvesting the livers. For the chemical

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analyses, liver samples were flash-frozen in liquid nitrogen and stored at -80 °C, and blood and food

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samples were stored at -20 °C. The samples were transferred to and stored at the Environmental

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Specimen Bank for Global Monitoring (es-BANK) at Ehime University, Japan.27

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Chemicals

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The authentic reference standards of tri- to penta-BPhs congeners (2,3,4-TBPh, 2,3,5-TBPh,

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2,3,6-TBPh, 2,4,5-TBPh, 2,4,6-TBPh, 3,4,5-TBPh, 2,3,4,5-TeBPh, 2,3,4,6-TeBPh, 2,3,5,6-TeBPh,

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and penta-BPh: PBPh) (structure shown in Figure 1) used for identification and quantification were

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obtained from AccuStandard, Inc. (New Haven, CT, USA).

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(2,4,6-TBPh, 2,3,4,6-TeBPh, and PBPh) were spiked as internal standards obtained from


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C-labeled tri- to penta-BPhs

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AccuStandard, Inc. (New Haven, CT, USA).

C-labeled BDE139 was added as a syringe spike

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obtained from Wellington Laboratories Inc. (Ontario, Canada).

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Measurement of BPhs in the Pet Blood and Food

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Samples (whole blood: 2–10 g; finely ground pet food: 10 g) spiked with

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standards were denatured with 6 M HCl and 2-propanol and then extracted with 50% methyl t-butyl

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ether (MTBE)/hexane. The clean-up and instrumental methods for BPhs followed our previously

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reported analytical methods for halogenated phenols.19,

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solution, BPhs were re-extracted, cleaned-up with deactivated silica-gel, and then derivatized with

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trimethylsilyldiazomethane. The methoxylated derivatives were further cleaned-up with activated

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silica-gel, gel-permeation chromatography, and then finally identified and quantified using gas

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chromatography–high-resolution mass spectrometry (GC–HRMS).

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C-labeled internal

Briefly, after partitioning into a KOH

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Quality Assurance and Quality Control

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BPhs were quantified using the isotope dilution method with the corresponding

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standards.29, 30 Recoveries for the 13C-labeled internal standards in pet blood were within the ranges

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of 15–91% (2,4,6-TBPh), 15–72% (2,3,4,6-TeBPh), and 19–76% (PBPh). Recoveries for the

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(2,3,4,6-TeBPh), and 25–56% (PBPh). Two of cat hematic samples were showed remarkably low

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C-internal

C-labeled internal standards in pet food were within the ranges of 29–98% (2,4,6-TBPh), 27–83%



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recoveries (15% for 2,4,6-TBPh; 15 and 17% for 2,3,4,6-TeBPh; 19 and 22% for PBPh) because of

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ion suppression due to the matrix contained within whole blood. However, these samples with high

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matrix effects were corrected for the influence of foreign substances by 13C-labeled internal standards

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in this study. Only 2,4,6-BPh was detected in the procedure blanks accompanying every five-sample

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batch, but the levels were only around 10% of those detected in the blood and food samples. Other

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details on QA/QC were described elsewhere.28

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Preparation of Cat Liver Microsomes and Analysis of Proteins and CYP Levels

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The preparation of the liver microsomes followed previously published methods.31 Briefly, 100-200

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mg of excised livers were homogenized in 5 volumes of cold homogenization buffer (50 mM

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Tris-HCl, 0.15 M KCl, pH 7.4-7.5) with a Teflon-glass homogenizer (10 passes). The homogenized

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liver samples were centrifuged for 10 min at 750 × g. After centrifugation, the nuclear pellet was

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removed and the supernatant was centrifuged at 12,000 × g for 10 min at 4 °C. The recovered

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supernatant was further centrifuged at 105,000 × g for 90 min at 4 °C. The microsomal pellet

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recovered from the centrifugation was resuspended in 1 volume of resuspension buffer (50 mM

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Tris-HCl, 1 mM EDTA, 1 mM DTT, 20% (v/v) glycerol, pH 7.4-7.5). An aliquot of each microsome

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fraction was used for the measurement of protein content using a bicinchoninic acid (BCA) Protein

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Assay Reagent (Pierce, Rockford, IL) and bovine serum albumin as a standard were used for the

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protein assay. Absorbance at 560 nm was measured using a multiwell plate reader (SpectraFluor Plus,


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Tecan Austria GmbH, Groedig, Austria).

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The level of CYPs in the cat liver microsomes was determined from the sodium dithionite-reduced

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CO difference spectrum at approximately 450 and 490 nm (91 mM−1cm−1 extinction coefficient) with

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the DU800 spectrophotometer (Beckman Coulter, Inc.). More details of the analytical procedures of

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BCA and CYP levels are provided in Mizukawa et al. (2016).19

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In Vitro Assay of Biotransformation of PBDEs

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The reaction mixture (1 mL final volume) contained the buffer (80 mM NaH2PO4, 6 mM MgCl2, 1

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mM Na2EDTA, pH 8.0), PBDE mixtures (BDE-47, BDE-99, and BDE-209) (purity >98%, 1 mg L-1

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each), and the microsomal suspension (200 pmol of CYPs). For the control sample, the reaction

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mixture contained only the buffer and the microsome. The mixture solution was preincubated at

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37 °C for 10 min and the CYP-dependent reaction was initiated by adding NADPH-regenerating

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solutions (50 µL of solution A and 10 µL of solution B) (BD Biosciences, NU, USA). The solution

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was incubated for 180 min in a shaking (90 rpm) water bath at 37 °C. The negative control reaction

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mixture contained the buffer, PBDE mixtures, and microsomes without the NADPH-regenerating

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solution. After incubation, the reaction was stopped by adding 1 mL of ice-cold methanol. The

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methods used for the analysis of BPhs and PBDEs in the reaction mixture are described in a previous

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subsection.

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Statistical Analysis

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The Mann-Whitney U-test was used to test the statistical significance of differences in the levels of

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BPhs between species. Spearman’s rank correlation coefficients were calculated to evaluate the

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relationship between the concentrations of BPhs and OH-/MeO-PBDEs levels showed in Mizukawa

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et al. (2016). 19 A p value of

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