Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Species- and Tissue-Specific Profiles of Polybrominated Diphenyl Ethers and Their Hydroxylated and Methoxylated Derivatives in Cats and Dogs Kei Nomiyama, Kohki Takaguchi, Hazuki Mizukawa, Yasuko Nagano, Tomoko Oshihoi, Susumu Nakatsu, Tatsuya Kunisue, and Shinsuke Tanabe Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Environmental Science & Technology
1
Research article
2
Species- and Tissue-Specific Profiles of Polybrominated Diphenyl Ethers and Their
3
Hydroxylated and Methoxylated Derivatives in Cats and Dogs
4 5
Kei Nomiyama†*, Kohki Takaguchi†, Hazuki Mizukawa‡, Yasuko Nagano†, Tomoko
6
Oshihoi†, Susumu Nakatsu§, Tatsuya Kunisue†, and Shinsuke Tanabe†
7 8
†
9
Matsuyama, Ehime 790-8577, Japan
Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,
10
‡
11
Sapporo 060-0818, Japan
12
§
13
Japan
Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku,
Nakatsu Veterinary Surgery, 2-2-5, Shorinjichonishi, Sakai-ku, Sakai, Osaka 590-0960,
14 15 16
Kei Nomiyama, Ph.D. (Correspondence)*
17
Center for Marine Environmental Studies (CMES), Ehime University,
18
Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
19
Tel: +81-89-927-8171
20
E-mail:
[email protected] 21 22 23 24 25
ACS Paragon Plus Environment
1
Environmental Science & Technology
Page 2 of 34
26
Abstract
27
The adverse effects of elevated polybrominated diphenyl ether (PBDE) levels, reported in the
28
blood of domestic dogs and cats, are considered to be of great concern. However, the tissue
29
distribution of PBDEs and their derivatives in these animals is poorly understood. This study
30
determined the concentrations and profiles of PBDEs, hydroxylated PBDEs (OH-PBDEs),
31
methoxylated PBDEs (MeO-PBDEs), and 2,4,6-tri-bromophenol (2,4,6-tri-BPh) in the blood,
32
livers, bile, and brains of dogs and cats in Japan. Higher tissue concentrations of PBDEs were
33
found in cats, with the dominant congener being BDE209. BDE207 was also predominant in
34
cat tissues, indicating that BDE207 was formed via BDE209 debromination. BDE47 was the
35
dominant congener in dog bile, implying a species-specific excretory capacity of the liver.
36
OH-PBDE and MeO-PBDE concentrations were several orders of magnitude higher in cat
37
tissues, with the dominant congener being 6OH-BDE47, possibly owing to their intake of
38
naturally occurring MeO-PBDEs in food, MeO-PBDE demethylation in the liver, and lack of
39
UDP-glucuronosyltransferase, UGT1A6. Relatively high concentrations of BDE209,
40
BDE207, 6OH-BDE47, 2′MeO-BDE68, and 2,4,6-tri-BPh were found in cat brains,
41
suggesting a passage through the blood–brain barrier. Thus, cats in Japan might be at a high
42
risk from PBDEs and their derivatives, particularly BDE209 and 6OH-BDE47.
43 44 45 46
Keywords Cat, Dog, PBDE, liver, brain, OH-PBDE, BDE209, 6OH-BDE47, Metabolic capacity
47 48 49 50
ACS Paragon Plus Environment
2
Page 3 of 34
51
Environmental Science & Technology
1. Introduction
52
Polybrominated diphenyl ethers (PBDEs) have become widespread contaminants of the
53
environment, humans, and wildlife because of their persistent and bioaccumulative
54
properties.1,2 The Stockholm Convention recently declared penta- and octa-BDE technical
55
mixtures as persistent organic pollutants.3,4 In Japan, technical tetra- and octa-BDE mixtures
56
were used as flame retardants until 1990 and 1999, respectively, and technical deca-BDE still
57
remains in use.5 In 2015, deca-BDE was recommended for inclusion in the Annex A list at
58
the 11th meeting of the Persistent Organic Pollutants Review Committee. Despite these
59
regulations, PBDEs likely continue to enter the environment through the disposal and
60
degradation of products containing PBDEs technical mixtures.
61
The detection of PBDE metabolites (hydroxylated PBDEs [OH-PBDEs] and bromophenols
62
[BPhs]) in the plasma of wild animals6–8 and human blood9,10 suggests that the
63
biotransformation of PBDEs occurs in the livers of animals.10–12 Structurally, OH-PBDEs and
64
BPhs resemble the thyroid hormone (TH) thyroxin and can bind to TH transport proteins
65
(e.g., transthyretin and thyroxine-binding globulin), which disrupt homeostasis.12–15 In
66
addition,
67
neurotoxicity.17,18 These studies suggest that the brain and liver are useful organs for
68
understanding the toxicokinetics of OH-PBDEs.
OH-PBDEs
reportedly
interrupt
oxidative
phosphorylation16
and
elicit
69
The presence of OH-PBDEs in concentrations higher than those of their parent PBDEs in
70
marine organisms has shown that OH-PBDEs and methoxylated PBDEs (MeO-PBDEs) may
71
be formed naturally by marine algae or cyanobacteria.19–21 Compared with PBDEs, MeO-
72
PBDEs have been found in various animals at higher concentrations.8,22,23 The demethylation
73
of MeO-PBDEs by cytochrome P450 (CYP) can result in the formation of OH-PBDEs. In
74
fact, the possibility that OH-PBDE congeners are formed by the demethylation of MeO-
75
PBDEs rather than by the metabolism of parent PBDEs in some species has been reported.24
ACS Paragon Plus Environment
3
Environmental Science & Technology
Page 4 of 34
76
2,4,6-tri-bromophenol (2,4,6-tri-BPh) is used commercially as a flame retardant and wood
77
preservative/fungicide with a worldwide.25 2,4,6-tri-BPh has been found in mussels and the
78
blubber of marine mammals.26
79
Domestic pets such as dogs and cats share living environments with humans. Therefore,
80
they are exposed to various contaminants, including PBDEs, in their immediate surroundings,
81
which raise concerns about health risks.27–29 In particular, recent studies have reported
82
elevated PBDE levels in the sera of cats.30–32 Moreover, evidence suggests that the main
83
routes of PBDE exposure in domestic cats are dietary intake and the ingestion of
84
contaminated house dust.31–33 Notably, compared with euthyroid cats, hyperthyroid cats have
85
higher serum concentrations of PBDE congeners (BDE99, BDE153, and BDE183), which
86
suggests that feline hyperthyroidism (FH) might be associated with increased exposure to
87
PBDEs.29 The number of cats diagnosed with FH has increased significantly during the last
88
three decades, and studies have suggested that the pathogenesis of FH involves exposure to
89
goitrogens, including PBDEs and phenolic metabolites such as OH-PBDEs.33-35
90
Compared with marine mammals, terrestrial carnivore species can have a higher metabolic
91
capacity for organohalogen compounds.36 Our recent study demonstrated that PBDE and OH-
92
PBDE levels in the blood of cats were higher than those of other carnivorous species.36 In
93
particular, the elevated levels of 6OH-BDE47 and 2′OH-BDE68 observed in the blood of cats
94
suggested that these OH-PBDE congeners formed via the demethylation of 6MeO-BDE47
95
and 2′MeO-BDE68, which occur naturally in seafood.33 Conversely, trace levels of 6OH-
96
BDE47 and 2′OH-BDE68 have been detected in the blood of dogs, which indicates that dogs
97
either metabolize OH-PBDE congeners more rapidly than cats or are exposed to much lower
98
levels of these natural compounds.36,37 Thus, among carnivorous species, cats might be at
99
high risk from 6OH-BDE47 and 2′OH-BDE68 exposure, and the metabolic capacities of
100
CYPs and binding affinities to proteins such as transthyretin (TTR) likely differ in dogs and
ACS Paragon Plus Environment
4
Page 5 of 34
Environmental Science & Technology
101
cats.33,36 Nevertheless, the residue levels and profiles of PBDEs, OH-PBDEs, and MeO-
102
PBDEs in tissues other than blood remain relatively unknown in dogs and cats.
103
Considering that the complex action of PBDEs and OH-PBDEs may be responsible for the
104
increased incidence of FH, further intensive studies are required to assess the toxicokinetics
105
of not only these parent compounds but also their derivatives in domestic animals. The
106
objective of this study was to determine the tissue-specific congener patterns of PBDEs and
107
their derivatives (OH-PBDEs MeO-PBDEs, and 2,4,6-tri-BPh) by analyzing the livers, blood,
108
bile, and brains of domestic dogs and cats.
109 110
2. Materials and Methods
111
2.1. Sample Collection
112
Blood, liver, and brain (cerebrum) samples from stray cats (n = 10; six males and four
113
females) and dogs (n = 10; five males and five females) found dead owing to traffic-related
114
trauma were collected between 2008 and 2011.33 Bile samples from stray cats (n = 5; four
115
males and one female) and dogs (n = 5; five males) were also collected. Although the
116
nutritional status and lipid content of the samples were not analyzed, there was no indication
117
that any of the animals was severely malnourished. The collected brain samples included a
118
small quantity of blood. These samples were transferred to the Environmental Specimen
119
Bank (es-BANK) for Global Monitoring (http://esbank-ehime.com/dnn/) at Ehime University
120
(Matsuyama, Japan) and stored at −25 °C until analysis.38 The characteristics of the analyzed
121
samples are shown in Table S1.
122 123
2.2. Chemicals
124
The authentic reference standards of the 11 PBDE congeners (BDE47, 99, 100, 153, 154,
125
183, 196, 197, 206, 207, and 209; ≥98% purity) were obtained from Wellington Laboratories
ACS Paragon Plus Environment
5
Environmental Science & Technology
Page 6 of 34
126
Inc. (Guelph, ON, Canada). MeO-PBDE congeners (≥98% purity) were obtained from
127
Wellington Laboratories Inc. and AccuStandard, Inc. (New Haven, CT, USA). 2,4,6-tri-BPh
128
(≥98% purity) was obtained from Wellington Laboratories Inc. The details of the MeO-
129
PBDE congeners used for identification and quantification are given in Table S2. 13C-labeled
130
tetra- and penta-brominated OH-PBDEs (6OH-BDE47, 6′OH-BDE99, 6′OH-BDE100; ≥99%
131
purity),
132
BDE28, BDE47, BDE99, BDE153, BDE154, BDE183, BDE196, BDE197, BDE206,
133
BDE207, BDE209; ≥99% purity) were purchased from Wellington Laboratories, Inc. as
134
internal standards.
13
C-labeled 2,4,6-tri-BPh (≥99% purity) and
13
C-labeled PBDEs (BDE3, BDE15,
135 136
2.3. Measurement of PBDEs and Their Metabolites
137
Analytical methods for measuring PBDEs, OH-PBDEs, MeO-PBDEs, and 2,4,6-tri-BPh
138
have been previously reported.8,39 Briefly, a whole blood sample (approximately 3–5 g) and
139
tissue samples (liver, bile, and brain; approximately 2.5 g) spiked with
140
standards were denatured with 6 M HCl and homogenized with 2-propanol and 50% methyl
141
t-butyl ether/hexane. After centrifugation, the organic phase was partitioned into neutral and
142
phenolic fractions using 1 M KOH in 50% ethanol/water. After the lipids in the neutral
143
fraction were removed using gel permeation chromatography (GPC), the GPC fraction
144
containing PBDEs and MeO-PBDEs was passed through an activated silica gel column. The
145
phenolic fraction was acidified with sulfuric acid and re-extracted twice with 50% methyl t-
146
butyl ether/hexane. The extracted solution containing OH-PBDEs and 2,4,6-tri-BPh was
147
passed through a column packed with deactivated silica gel (5% H2O deactivated) and
148
derivatized overnight using trimethylsilyldiazomethane. The derivatized solution was passed
149
through an activated silica gel column after lipid removal with GPC, and the MeO-PBDEs
150
were eluted with 10% dichloromethane/hexane. A gas chromatograph (6890 series, Agilent
ACS Paragon Plus Environment
13
C-labeled internal
6
Page 7 of 34
Environmental Science & Technology
151
technologies) coupled to a high-resolution (>10,000) mass spectrometer (JMS-800D, JEOL)
152
was used to identify and quantify the target of low concentration of PBDEs, MeO-PBDEs
153
and 2,4,6-tri-BPh. Highly brominated PBDEs (octa- to deca-BDEs) were quantified using the
154
gas chromatograph (6890 series, Agilent technologies) and mass spectrometer (5973N,
155
Agilent technologies) in electron impact and selected ion monitoring mode.40 Details are
156
described in SI.
157 158
2.4. Statistical Analyses
159
The Mann–Whitney U-test was used to test significant differences. Spearman’s rank
160
correlation coefficients were calculated to evaluate the strength of the relation between
161
PBDE, MeO-PBDE, OH-PBDE, and 2,4,6-tri-BPh concentrations in the animals. If
162
concentrations of organobromine compounds were below the limit of quantification (LOQ),
163
the values of the half of LOQ concentrations were used for statistical analyses. A p value of
164
