Organohalogen Compounds in Pet Dog and Cat: Do Pets

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Organohalogen compounds in pet dog and cat: Do pets biotransform natural brominated products in food to harmful hydroxlated substances? Hazuki Mizukawa, Kei Nomiyama, Susumu Nakatsu, Hisato Iwata, Jean Yoo, Akira Kubota, Miyuki Yamamoto, Mayumi Ishizuka, Yoshinori Ikenaka, Shouta MM Nakayama, Tatsuya Kunisue, and Shinsuke Tanabe Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04216 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015

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Environmental Science & Technology 1

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Organohalogen compounds in pet dog and cat: Do pets biotransform natural brominated

2

products in food to harmful hydroxlated substances?

3 4

Hazuki Mizukawa†1, Kei Nomiyama†*, Susumu Nakatsu‡, Hisato Iwata†, Jean Yoo†, Akira Kubota§,

5

Miyuki Yamamoto†, Mayumi Ishizukaǁ, Yoshinori Ikenakaǁ, Shouta M. M. Nakayamaǁ Tatsuya

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Kunisue†, and Shinsuke Tanabe†

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†

9

Matsuyama, Ehime 790-8577, Japan

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

10

‡

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

11

§

Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and

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Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan

13

ǁ

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Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan

Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18,

15 16 17

*Address correspondence to:

18

Kei Nomiyama, Ph.D.

19

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama, Ehime 790-8577, Japan

20 21

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

22

E-mail: [email protected]

23 24

1

25

Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan

Present address: Department of Environmental Veterinary Science, Graduate School of Veterinary

26

ACS Paragon Plus Environment

Environmental Science & Technology

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Abstract

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There are growing concerns about the increase in hyperthyroidism in pet cats due to exposure to

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organohalogen contaminants and their hydroxylated metabolites. This study investigated the blood

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contaminants polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) and

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their hydroxylated and methoxylated derivatives (OH-PCBs, OH-PBDEs, and MeO-PBDEs), in pet

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dogs and cats. We also measured the residue levels of these compounds in commercially available

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pet foods. Chemical analyses of PCBs and OH-PCBs showed that the OH-PCB levels were one to

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two orders of magnitude lower in cat and dog food products than in their blood, suggesting that the

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origin of OH-PCBs in pet dogs and cats is PCBs ingested with their food. The major congeners of

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OH-/MeO-PBDEs identified in both pet food products and blood were natural products

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(6OH-/MeO-BDE47 and 2′OH-/MeO-BDE68) from marine organisms. In particular, higher

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concentrations of 6OH-BDE47 than 2′OH-BDE68 and two MeO-PBDE congeners were observed

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in the cat blood, although MeO-BDEs were dominant in cat foods, suggesting the efficient

40

biotransformation of 6OH-BDE47 from 6MeO-BDE47 in cats.

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We performed in vitro demethylation experiments to confirm the biotransformation of

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MeO-PBDEs to OH-PBDEs using liver microsomes. The results showed that 6MeO-BDE47 and

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2′MeO-BDE68 were demethylated to 6OH-BDE47 and 2′OH-BDE68 in both animals, whereas no

44

hydroxylated metabolite from BDE47 was detected. The present study suggests that pet cats are

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exposed to MeO-PBDEs through cat food products containing fish flavors and that the OH-PBDEs

46

in cat blood are derived from the CYP-dependent demethylation of naturally occurring MeO-PBDE

47

congeners, not from the hydroxylation of PBDEs.

48 49 50 51 52


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Introduction

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Organohalogen compounds such as polychlorinated biphenyls (PCBs) and polybrominated

56

diphenyl ethers (PBDEs) are widely used in industry. Because of their persistence and high

57

bioaccumulative potency, PCBs and PBDEs have been detected in both animal species and humans

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at significant levels.1,2 These halogenated contaminants adversely affect the endocrine system and

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neurodevelopment.3 Moreover, hydroxylated metabolites of PCBs (OH-PCBs) and PBDEs

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(OH-PBDEs) disrupt thyroid hormone (TH) homeostasis.4 OH-PCBs and OH-PBDEs are formed in

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the phase I metabolic pathway, which is mediated by the cytochrome P450 (CYP) monooxygenase

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system.5,6 OH-PBDE congeners such as 6OH-BDE47 and 2′OH-BDE68 are produced by marine

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sponges, cyanobacteria, and algae.7-9 Studies of the Japanese medaka (Oryzias latipes) have

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reported that OH-PBDEs are formed by the demethylation of the methoxylated PBDEs

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(MeO-PBDEs) which occur naturally in the marine organisms mentioned above.10 These

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hydroxylated metabolites are structurally similar to thyroxin (T4), thus bind to the TH transport

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protein transthyretin (TTR) with a much higher affinity than their parent compounds. The effects of

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OH-PCBs and OH-PBDEs are thus of concern, because they have been detected in the tissues of a

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variety of animals.11-14

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Pet dogs and cats might be exposed to environmental contaminants including PCBs and

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PBDEs.15-18 One previous study investigated organochlorine compound residues in cats and dogs

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from a wide area of Southern Italy and reported that PCB concentrations were higher in cats than in

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dogs.15 In addition, concentrations of PBDEs in the serum of the dogs were significantly lesser than

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those measured in the serum of cats in the USA.16 These results suggested that differences of size

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class, dietary exposure, and/or xenobiotic metabolizing systems exist between the species. Other

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studies have detected higher levels of PBDEs in the sera of pet cats than in the sera of humans.18-20

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Moreover, evidence suggests that the main routes of exposure to PBDEs for pet cats are diet and

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ingested contaminated house dust.18,20,21 Cats are expected to have higher exposure to PBDE



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because of increased intake of house dust from their grooming behavior.20,22 Several reports have

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hypothesized that increases in feline hyperthyroidism (FH) might be associated with increased

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exposure to PBDEs.18,19 A more recent study has suggested that hyperthyroid cats have higher

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serum concentrations of PBDEs (BDE99, BDE153, and BDE183) and CB153 than euthyroid cats.23

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The number of cats diagnosed with FH has increased significantly over the last three decades, and

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the multiple risk factors for FH suggest that its pathogenesis involves exposure to goitrogens,

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including PBDEs.24,25 The increased incidence of FH might be linked to the incorporation of

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phenolic metabolites such as OH-PCBs and OH-PBDEs.19,20 Conversely, hyperthyroidism in dogs is

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very rare and is a iatrogenic disease caused by the medical treatment of hypothyroidism, for

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example, by the excess administration of an ergogenic thyroid hormone.

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Terrestrial carnivorous species have a higher metabolic capacity for organohalogen compounds

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than marine mammals. Moreover, the levels of PBDEs and OH-PBDEs measured in the blood of

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cats have been shown to be higher than those of other carnivorous species.26,27 In particular, high

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levels of 6OH-BDE47 and 2′OH-BDE68 were found in the blood of cats, suggesting the ingestion

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of the natural compounds from seafood. Conversely, low concentrations of these natural compounds

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were found in the blood of dogs. These results suggest either that dogs metabolize the natural

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compounds more rapidly than cats or that dogs are exposed to much lower levels of the

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compounds.26 Our previous study suggested that the different residue levels of these compounds

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found among carnivorous species indicate a high risk of 6OH-BDE47 and 2′OH-BDE68 in cats26

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and that the metabolic capacities and binding affinities with specific proteins such as TTR are

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different in dogs and cats. However, there have been no reports of the biotransformation of

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organohalogen compounds to hydroxylated metabolites in dogs and cats. In addition, our previous

101

study showed that lower-chlorinated OH-PCB congeners (3-5 Cl) were predominant (more than

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80% to the total OH-PCBs) in cat blood, whereas higher-chlorinated OH-PCBs (6–8 Cl) were major

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congeners in the blood of other carnivorous species.26 These findings suggest that halogenated

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phenolic compounds are preferentially retained in the blood of cats, because they do not undergo


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robust phase II conjugation. This hypothesis is consistent with the fact that the

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UDP-glucuronosyltransferase UGT1A6 is lacking in cats.28 Nevertheless, the differences between

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the activities of enzymes that metabolize PCBs and PBDEs in dogs and cats remain unknown, and

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the data on exposure levels to these contaminants through pet food products remain insufficient.

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Considering that the increased incidence of FH may be responsible for the incorporation of phenolic

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compounds such as OH-PCBs and OH-PBDEs, more intensive study is necessary to assess the

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exposure and residue levels of these hydroxylated metabolites and to investigate their formation

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processes in these animals. However, only limited information is available on the levels of the

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metabolites of PCBs and PBDEs in pet animals and their food.16,23

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The present study determined the levels and accumulation patterns of PCBs, PBDEs, and their

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metabolites (OH-PCBs, OH-PBDEs, and MeO-PBDEs) in the blood of pet cats and dogs collected

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from a veterinary hospital in Japan. To estimate the exposure routes to these chemicals, we

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determined the levels of dietary exposure of these pets to PCBs, PBDEs, and their derivatives from

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representative samples of dry and wet pet food products. In addition, we conducted in vitro

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demethylation experiments to confirm the biotransformation of MeO-PBDEs to OH-PBDEs in the

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livers of dogs and cats. Finally, we compared the biotransformation capacity of PCBs and PBDEs in

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dogs and cats.

122 123 124

Experimental section

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Sample Collection

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Blood samples from pet dogs (n = 17) and cats (n = 11) were collected at the Nakatsu Veterinary

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Surgery in Osaka and the Tao Veterinary Hospital in Hiroshima, Japan, during 2009–2012. The pets

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were brought to the veterinary hospitals for clinical treatments such as surgical procedures (for

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lymphoma and pyometra, neutering etc.), but excluding FH. The owners of the pets completed a

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questionnaire, providing information about age, sex, weight, housing conditions, eating habits (dry



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or wet food), and housing environment (indoors or outdoors) (Table S1). Commercial dry and wet

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pet food products of the most popular brands in Japan were purchased from Japanese pet shops in

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2010. The details of the pet food samples (n = 16) are presented in Table S2.

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The pooled liver microsome from ten healthy beagle dogs used for the in vitro demethylation

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experiments was purchased from Life Technologies (Carlsbad, CA, USA). We collected fresh liver

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samples (within 30 min of the donor’s death) from three domestic cats, with the cooperation of

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Nakatsu Veterinary Surgery in Osaka, Japan (Table S3). These cats were euthanized using

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pentobarbital sodium, because of incurable (and painful) cases or diseases. Owners provided

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consent for the harvesting of the livers. For the chemical analyses, liver samples were flash-frozen

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in liquid nitrogen and stored at −80 °C and blood and food samples were stored at −20 °C. The

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samples were transferred to and stored at the Environmental Specimen Bank for Global Monitoring

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(es-BANK) at Ehime University, Japan.29

143 144

Chemicals

145

The standards for the 62 PCB and 52 OH-PCB (methoxylated derivatives; MeO-PCBs)

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congeners are described in the Supporting Information (SI) and Table S4. The standards for the 9

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PBDE congeners (BDE47, 99, 100, 153, 154, 183, 196, 197, 206, 207, and 209) were obtained from

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Wellington Laboratories Inc. (Guelph, ON, Canada). MeO-PBDE congeners (methoxylated

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derivatives; MeO-PBDEs) were obtained from Wellington Laboratories Inc. (Guelph, ON, Canada),

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Accu Standard, Inc. (New Haven, CT), and Cambridge Isotope Laboratories Inc., USA. Details of

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the nine PBDE and 28 MeO-PBDE (OH-PBDEs derivatives) congeners are presented in the SI and

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in Table S5. 13C-labeled tri- to hepta-chlorinated OH-PCBs (4OH-CB29, 4ÓH-CB61, 4OH-CB79,

153

4ÓH-CB120, 4OH-CB107, 4ÓH-CB159, 4OH-CB146, 4ÓH-CB172, and 4OH-CB187),

154

13

155

6ÓH-BDE100),

156

CB156, CB157, CB167, CB170, CB178, CB180, CB189, CB194, CB202, CB206, and CB208), and

C-labeled

tetra-

and

penta-brominated

OH-PBDEs

(6OH-BDE47,

6ÓH-BDE99,

and

13

C-labeled PCBs (CB28, CB52, CB95, CB101, CB105, CB118, CB138, CB153,


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157

13

158

BDE196, BDE197, BDE206, BDE207, and BDE209) were spiked as internal standards obtained

159

from Wellington Laboratories Inc. (Guelph, ON, Canada).

C-labeled PBDEs (BDE3, BDE15, BDE28, BDE47, BDE99, BDE153, BDE154, BDE183,

160 161

Analysis of PCBs, PBDEs, OH-PCBs, OH-PBDEs, and MeO-PBDEs in the Blood

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The analytical methods for PCBs, OH-PCBs, PBDEs, OH-PBDEs, and MeO-PBDEs have been

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reported elsewhere.30,31 Briefly, a whole blood sample (approximately 3–5 g) in which 13C-labeled

164

internal standards were spiked was denatured with 6 M HCl and homogenized with 2-propanol and

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50% methyl t-butyl ether (MTBE)/hexane. After centrifugation, the organic phase was partitioned

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into neutral and phenolic fractions using 1 M KOH in 50% ethanol/water. After lipids in the neutral

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fraction had been removed using gel permeation chromatography (GPC), the GPC fraction

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containing PCBs, PBDEs, and MeO-PBDEs was passed through an activated silica gel column. The

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phenolic fraction was acidified with sulfuric acid and re-extracted twice with 50% MTBE/hexane.

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The extracted solution containing OH-PCBs and OH-PBDEs was passed through a column packed

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with inactivated silica gel (5% H2O deactivated). Moreover, OH-PCBs and OH-PBDEs were eluted

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with 50% DCM/hexane (100 mL), concentrated, dissolved in hexane (1 mL), and then derivatized

173

to

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trimethylsilyldiazomethane. The derivatized solution was passed through an activated silica gel

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column after the lipids had been removed by GPC, and MeO-PCBs and MeO-PBDEs were eluted

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with 10% DCM/hexane. A gas chromatograph (GC, 6890 series, Agilent) coupled to a

177

high-resolution (>10,000) mass spectrometer (HRMS, JMS-800D, JEOL) was used to identify and

178

quantify the target organohalogen compounds. Highly brominated PBDEs (Octa–deca BDEs) were

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quantified using a GC (6890 series, Agilent)/MS (5973N, Agilent).32 Electron impact and selected

180

ion monitoring mode (EI-SIM) was used for the GC/MS analyses.

methylated

compounds

(MeO-PCBs

and

MeO-PBDEs)

overnight

181 182

Analysis of PCBs, PBDEs, OH-PCBs, OH-PBDEs, and MeO-PBDEs in Pet Food Products


using


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183

The contents of the organohalogen compounds in the pet food samples were analyzed using a

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previously reported method. 30,31 A pet food sample (approximately 10 g) was crushed using a

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mortar and homogenizer and then extracted with 6 M HCl, 2-propanol, and 50% MTBE/hexane in

186

the same way as the blood samples. The details are described in the SI.

187 188 189 190

Quality Assurance and Quality Control All organohalogen compounds were quantified using the isotope dilution method with the corresponding 13C12-internal standards.26,31 The details of QA/QC are given in the SI.

191 192

Preparation of Cat Liver Microsomes and Analysis of Proteins

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The preparation of the liver microsomes followed previously published methods.33,34 Briefly,

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100–200 mg of excised livers was homogenized in 5 vol. of cold homogenization buffer (50 mM

195

Tris–HCl, 0.15 M KCl, pH 7.4–7.5) with a Teflon-glass homogenizer (10 passes), and the

196

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

197

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

198

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

199

pellet recovered from the centrifugation was resuspended in 1 vol. of resuspension buffer (50 mM

200

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

201

microsome fraction was used for the measurement of protein content using a bicinchoninic acid

202

(BCA) assay kit. More details are provided in the SI.

203 204

Analysis of CYP Levels and Enzyme Activities

205

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

206

dithionite-reduced CO difference spectrum at approximately 450 and 490 nm (91 mM−1 cm−1

207

extinction coefficient). The CYP spectra were analyzed as described in the SI. Measurements of

208

alkoxyresorufin O-dealkylase (AROD) activities in microsomal fractions and western blotting were


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performed with minor modifications of published methods.33 Details are provided in the SI.

210 211

In Vitro Assay of Biotransformation of PBDEs and MeO-PBDEs

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

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1 mM Na2EDTA, pH 8.0), 10 ng of BDE47 (purity >98%) or a mixture of 6MeO-BDE47 (>98%)

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and 2′MeO-BDE68 (>98%) in 2% DMSO, and the microsomal suspension (200 pmol of CYPs).

215

For the control sample, the reaction mixture contained only the buffer and the microsome. The

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mixture solution was preincubated at 37 °C for 10 min, and the CYP-dependent reaction was

217

initiated by adding NADPH-regenerating solutions (50 µL of solution A and 10 µL of solution B)

218

(BD Biosciences, NU, USA). The solution was incubated for 180 min in a shaking (90 rpm) water

219

bath at 37 °C. The negative control reaction mixture contained the buffer, BDE47 or MeO-PBDEs,

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and microsomes without the NADPH-regenerating solution. After incubation, the reaction was

221

stopped by adding 1 mL of ice-cold methanol. All assays were performed in triplicate. The methods

222

used for the analysis of PBDEs, OH-PBDEs, and MeO-PBDEs in the reaction mixture have been

223

reported elsewhere.30,31

224 225

Statistical Analysis

226

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

227

levels of target compounds between species. Spearman’s rank correlation coefficients were

228

calculated to evaluate the relationship between the concentrations of PCBs, OH-PCBs, PBDEs,

229

OH-PBDEs, and MeO-PBDEs in each species. A p value of Cat 2 > Cat 3) was consistent with that

379

of AROD activities (Table 2). These results indicate the preferential formation of 6OH-BDE47 from

380

6MeO-BDE47 by CYP catalytic activities, and support the observation of higher 6OH-BDE47

381

levels in the blood of pet cats (Table 2), compared with 2′OH-BDE68. This may also be explained

382

by the binding affinity of the TH transport protein for 6OH-BDE47 in the blood,45 and by the weak

383

ability to further metabolize this compound in the liver via a phase II conjugation reaction.46 The

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toxicological implications of cat exposure to 6OH-BDE47 remain unknown. However,

385

6OH-BDE47 is of particular interest because it triggers a variety of toxic effects such as

386

interruption of oxidative phosphorylation47 and inhibition of estradiol-sulfotransferase45 and is

387

neurotoxic48 in exposed wildlife and humans. A recent study showed that the hydroxylated

388

metabolite 6OH-BDE47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release

389

than the parent compound BDE47. This result suggests that bioactivation by metabolism adds

390

considerably to the neurotoxic potential of PBDEs.48 Further investigation is necessary to determine



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whether the accumulation of 6OH-BDE47 is associated with neurotoxicity and TH homeostasis in

392

cats.

393

In the dog microsome, 2′MeO-BDE68 was mostly demethylated to 2′OH-BDE68 (95%) and the

394

production rate of 6OH-BDE47 (44%) was also higher than the rate observed in the cat microsomes

395

(Figure 3). These results indicate that dogs have a higher MeO-PBDE demethylation capacity than

396

cats. However, 2′OH-BDE68 and 6OH-BDE47 were undetectable in the blood of pet dogs (Table 1

397

and Table S9). The low levels of MeO-BDEs in dog foods may be a contributing factor (Table S10);

398

and may also be attributed to the efficient conjugation metabolism of these OH-BDEs in dogs

399

because of their high phase II enzymatic activity.46 The differences in CYP-mediated demethylation

400

of MeO-BDEs to OH-BDEs may influence the levels of OH-BDEs in different mammalian

401

species.44 To our knowledge, no data are available on the biotransformation of 2′MeO-BDE68 to

402

2′OH-BDE68 by in vitro and in vivo assays, but a recent in vivo study using rainbow trout suggested

403

that the demethylation of 6MeO-BDE47 to 6OH-BDE47 might be mainly catalyzed by a member of

404

the CYP2 family.49

405

Using the same in vitro assays, we also investigated whether 6OH-BDE47 is formed by the

406

hydroxylation of BDE47. After BDE47 was added to the cat- and dog-liver microsomes and

407

incubated for 180 min, no hydroxylated metabolites of BDE47, including 6OH-BDE47, were

408

detected in this study (data not shown). However, several previous studies on BDE47 metabolism

409

have reported the detection of OH-PBDEs.45,50,51 The level of PBDE used for exposure in those

410

experiments was expressed as pg/g wet weight range, but OH-PBDEs were detected at

Organohalogen Compounds in Pet Dog and Cat: Do Pets

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