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Toxic identification and evaluation of androgen receptor antagonistic activities in acid-treated liver extracts of high-trophic level wild animals from Japan Kentaro Misaki, Go Suzuki, Nguyen Minh Tue, Shin Takahashi, Masayuki Someya, Hidetaka Takigami, Yuko Tajima, Tadasu Yamada, Masao Amano, Tomohiko Isobe, and Shinsuke Tanabe Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02288 • Publication Date (Web): 30 Aug 2015 Downloaded from http://pubs.acs.org on September 7, 2015

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Environmental Science & Technology

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Toxic identification and evaluation of androgen receptor antagonistic activities in acid-treated

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liver extracts of high-trophic level wild animals from Japan

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1,2

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Kentaro Misaki*, 3Go Suzuki, 1Nguyen Minh Tue, 1Shin Takahashi, 1Masayuki Someya,

Hidetaka Takigami, 4Yuko Tajima, 4Tadasu K. Yamada, 5Masao Amano, 1Tomohiko Isobe, 1

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Shinsuke Tanabe

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790-8577, Ehime, Japan

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2

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3

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

School of nursing, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan

Center for Material Cycles and Waste Management Research, National Institute for Environmental

Studies (NIES), Onogawa 16-2, Tsukuba 305-8506, Ibaraki, Japan

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4

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Japan

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5

National Museum of Nature and Science, Hyakunin-cho 3-23-1, Shinjuku-ku, Tokyo 169-0073,

Faculty of Fisheries, Nagasaki University, Bunkyo-cho 1-14, Nagasaki 852-8521, Japan

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*

Corresponding author: School of nursing, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka ACS Paragon Plus Environment


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422-8526,

Japan,

Telephone:

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[email protected]

+81-54-264-5462.

Fax:

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+81-54-264-5462.

E-mail:

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ABSTRACT

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Sulfuric acid-treated liver extracts of representative high-trophic level Japanese animals were analyzed

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by toxic identification and evaluation (TIE) with Chemically Activated Luciferase eXpression (CALUX)

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and chemical analysis to elucidate androgen receptor (AR) antagonistic activities and potential

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contributions of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). The activities

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were detected in striped dolphins (n = 5), Stejneger’s beaked whales (n = 6), golden eagle (n = 1), and

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Steller’s sea eagle (n = 1) with CALUX-flutamide equivalents (FluEQs) as follow: 38 (20-52), 47

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(21-96), 5.0, and 80 µg FluEQ/g-lipid, respectively. The AR antagonism was detected in limited number

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of specimens at lower levels for finless porpoise, raccoon dog, and common cormorant. Theoretical

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activities (Theo-FluEQs) were calculated using the concentration of OCPs and PCBs and their

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IC25-based relative potency (REP) values. These total contribution to CALUX-FluEQ was 126%, 84%,

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53%, 55%, and 44% for striped dolphin, Steller’s sea eagle, Stejneger’s beaked whale, finless porpoise,

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and golden eagle, respectively, and the main contributor was p,p′-DDE. However, most of the activities

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for raccoon dog (7.6%) and common cormorant (17%) could not be explained by OCPs and PCBs. This

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suggests other unknown compounds could function as AR antagonists in these terrestrial species.

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Keywords: AR antagonistic activity, TIE, high-trophic level wild animals, OCPs, PCBs, p,p′-DDE,

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AR-CALUX assay

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INTRODUCTION

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The release of large amounts of chemicals, such as organohalogen compounds, into the

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environment cause elevated levels of chemicals to accumulate in the biota. 1-7 Organochlorine pesticides

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(OCPs) and polychlorinated biphenyls (PCBs) were banned several decades ago and these chemicals are

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still found in various species. 8,9 Numerous reports have revealed that newer persistent organic pollutants

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(POPs) such as brominated flame retardants (BFRs)

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also accumulating in the biota. Adverse effects such as mass mortality, impaired neurodevelopment as

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well as immune and reproductive anomalies are associated with some of these environmental

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contaminants. 1,2,11

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5,9-11

and perfluorinated compounds (PFCs)

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are

One of the common observed effects of environmental contaminants in wildlife is feminization, 1-4,6

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most frequently reported between the 1980s and the 1990s.

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associated with reduced penis size of male alligators (Alligator mississippiensis) in Lake Apopka.

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addition, p,p′-DDE and PCBs concentrations were inversely correlated with blood testosterone levels of

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Dall’s porpoises (Phocoenoides dalli). 4 The disruptive effects of contaminants on nuclear receptors such

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as estrogen receptors (ERs), androgen receptors (ARs), and aryl hydrocarbon receptors (AhRs) may be

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associated with feminization.

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and effects of estrogen-like contaminants (ER agonists).

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environmental contaminants on ARs are limited

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several fungicides, germicides, flame retardants and pharmaceuticals as AR antagonists has been

13-15

For instance, p,p′-DDE has been 3

In

Many studies have been conducted on the environmental occurrence

3,4,7

2,13,14

However, the studies for the risk of

though, in fish and river water, the profiling of

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7,16

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reported.

The organochlorine compounds such as OCPs and PCB congeners have been found as AR

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antagonists,

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function, and sex-steroid-mediated behaviors in experimental male animals. 18-20

17

and were reported to exert detrimental effects on development, reproductive system

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The availability of bioassays allows evaluation of toxicological risk for animals related to the

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effects on receptor-specific endpoints caused by the mixture of various chemicals accumulated in their

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body. In our previous study, persistent (sulfuric acid-resistant) compounds in pooled tissue extracts from

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several high-trophic level animals were found to exert several endocrine-disrupting activities in toxic

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identification and evaluation (TIE) using a panel of cell-based Chemically Activated Luciferase

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eXpression (CALUX) assay.

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antagonism with p,p′-DDE contributing to 59% of this activity.

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assessments for AR antagonists in other animal species need to be investigated in relation with their

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accumulation levels/patterns.

21

The pooled Baikal seal (Phoca sibirica) extracts showed significant AR 22

These results suggest that risk

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In the present study, the occurrence of persistent AR antagonists in various high trophic level

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species, including additional cetaceans and raptors, was investigated. Sulfuric acid-treated liver extracts

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from seven Japanese animal species (striped dolphin [Stenella coeruleoalba], Stejneger’s beaked whale

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[Mesoplodon stejnegeri], finless porpoise [Neophocaena phocaenoides], raccoon dog [Nyctereutes

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procynoides], common cormorant [Phalacrocorax carbo], golden eagle [Aquila chrysaetos], and

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Steller’s sea eagle [Haliaeetus pelagicus]) were investigated using AR CALUX assay as well as

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chemical analysis in view of (a) total in vitro AR antagonist activity, (b) OCPs and PCBs concentrations,

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and (c) OCPs and PCBs contribution to the AR antagonist activity. ACS Paragon Plus Environment


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MATERIALS AND METHODS

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Chemicals

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OCPs dichlorodiphenyltrichloroethanes (DDTs), (p,p′-DDT, p,p′-DDD, p,p′-DDE) and chlordanes

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(CHLs), (cis-chlordane, trans- and cis-nonachlor) as well as hexachlorocyclohexanes (HCHs), (α-, β-,

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and, γ-isomers), hexachlorobenzene (HCB), and PCB congeners (PCB99, -118, -128, -138, -149, -153,

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-180, -183, -187) were used in the bioassays and were supplied by AccuStandard, Inc. (New Haven, CT,

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USA). Trans-chlordane, oxychlordane, and mirex were also used in the bioassays and were supplied by

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Sigma-Aldrich Japan G.K. (Tokyo, Japan), Crescent Chemical Co., Inc. (Islandia, NY, USA), and Wako

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Pure Chemical Ind., Ltd. (Osaka, Japan), respectively. These OCPs and PCB congeners are

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organochlorine compounds commonly reported to accumulate in various wild animals and can

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contribute as AR antagonistic candidates.

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Samples

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Liver tissues were obtained from male specimens of the following Japanese wild animals: striped

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dolphins (n = 5) from Seto Inland Sea in 2003, 9 Stejneger’s beaked whales (n = 6) from Japan Sea in

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2000–2003, finless porpoises from (n = 5) Omura Bay in 2005–2007,

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Kanagawa in 2001,

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Iwate in 1993, and Steller’s sea eagles (n = 1) from Hokkaido in 1999 (Fig. 1, Table S1). All specimens

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consisted of stranded marine mammals, common cormorants shot as part of a culling programme, and

10

5

raccoon dogs (n = 5) from

common cormorants (n = 6) from Lake Biwa in 2003, golden eagle (n = 1) from


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other animals died from natural or accidental causes. The samples were stored in the Environmental

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Specimen Bank (es-BANK) at Ehime University (Matsuyama, Ehime, Japan) at −25 ºC.

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Pretreatment of Samples

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For each species, 11 g of liver sample was freeze-dried and extracted with acetone-hexane (1:1, v/v)

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in a high speed solvent extractor (SE-100, Mitsubishi Chemical Analytech. Co., Ltd., Yokkaichi, Japan).

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The crude extract was solvent-exchanged with 10 mL of hexane and 4 mL of the solution was treated

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with sulfuric acid silica gel chromatography. Then the solvent was evaporated with a Kuderna-Danish

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concentrator and nitrogen gas. One-quarter of the concentrated solution was stored in 200 µL of hexane

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at 4 ºC until OCPs analysis while the rest of the concentrated solution was solvent-exchanged into 100

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µL of dimethyl sulfoxide (DMSO) and stored at 4 ºC until the CALUX assay. For PCBs analysis, 5 mL

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of crude extract was treated using sulfuric-acid-silica gel chromatography, gel permeation

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chromatography (GPC), and silica gel chromatography as reported previously. 5,9

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Chemical Analysis

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Nineteen OCPs (p,p′-DDT, p,p′-DDD, p,p′-DDE, o,p′-DDT, o,p′-DDD, o,p′-DDE), CHLs (trans-

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and cis-chlordane, oxychlordane, trans- and cis-nonachlor), HCHs (α-, β-, γ-, and δ-HCHs), HCB,

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methoxychlor, heptachlor, and mirex were analyzed with a gas chromatograph (GC 6890) interfaced

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with a mass-selective detector (5973N, Agilent Tech., Inc., Santa Clara, CA, USA). p-Terphenyl-d14 (0.2

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µg) was added to the pretreated extract as a syringe spike before injection. The GC column was heated ACS Paragon Plus Environment


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for 70 ºC for 0–1 min, 70–180 ºC for 1–6.5 min, 180–260 ºC for 6.5–22.5 min, 260–300 ºC for

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22.5–24.1 min, and 300 ºC for 24.1–31.1 min. Each sample injection volume (2 µL) was separated on a

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DB-17 ms capillary column using 30 m × 0.25 mm i.d. × 0.25 µm film thickness (Agilent J&W, Agilent

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Tech., Inc.). Electron impact-selected ion monitoring (EI-SIM) with constant ionization energy of 70 eV

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was used to analyze mass spectrometry. The quantification ion and reference ion for each OCP were

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p,p′-, o,p′-DDT (235.0, 237.0); p,p′-, o,p′-DDD (235.0, 237.0); p,p′-, o,p′-DDE (318.0, 316.0); trans-,

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cis-chlordane (372.9, 271.8); oxychlordane (184.9, 386.8); trans-, cis-nonachlor (408.8, 406.8); HCHs

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(181.0, 218.9); HCB (283.9, 285.9); methoxychlor (227.1, 228.1); heptachlor (271.8, 336.9); and mirex

149

(236.8, 271.8), respectively. For Stejneger’s beaked whale, finless porpoise, common cormorant, golden

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eagle, and Steller’s sea eagle extracts, the limits of detection (LODs) were determined, based on the

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blank concentration, the extraction and concentration factors, and sample volume (mean LOD values

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and ranges of the trophic species were 18 (8.5–28), 16 (8.3–29), 12 (10–14), 26, and 18 ng/g-lipid,

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respectively). The analyses of PCBs were conducted using a GC 6890–MS 5973N system, which is

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described in detail elsewhere.

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raccoon dog samples were previously reported. 5,10

5,9

The analytical results of OCPs and PCBs for the striped dolphin and

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AR CALUX Assay

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AR antagonist activity was evaluated according to a previously described method as AR-CALUX

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assay, which used U2OS-luc cells (human osteoblast cells transfected with human AR) developed by

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BioDetection Systems b.v. (Amsterdam, the Netherlands). 21-25 This method is different from the Chinese ACS Paragon Plus Environment

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17,26-29

or human prostate cancer cell

30-33

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hamster ovary (CHO)

AR-activated assays because it is

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sensitive to androgenic hormones and has little or no inherent receptor activity so crosstalk is minimal.

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Cells were maintained in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12

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(DMEM/F12) medium supplemented with 7.5% fetal bovine serum (FBS) and incubated at 37 °C in a

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humidified atmosphere containing 5% CO2. Cells were plated for 1 day on 96-well microplates. Then

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the cells were exposed for 24 h to DMEM/F12 medium (without phenol red) supplemented with 4.9%

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dextran-coated charcoal-treated FBS, which contained the samples or chemicals in the presence of an

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EC50 level (100 pM) of dihydrotestosterone (DHT). The medium was then removed and the cells were

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lysed. Luciferin solution was added and the luminescence intensity was measured with a luminometer

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(ARVO™ X3, PerkinElmer, Waltham, MA, USA). Sigmoid dose–response curves for the chemicals

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were fitted to the Hill equation (Equation 1) using SigmaPlot 11.0 (Systat Software Inc., San Jose, CA,

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USA):

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y = min + (max - min)/(1 + (a1/x)a2)

[1]

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where y = measured luciferase induction level; x = concentration; min = luciferase induction level at

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maximum inhibition by the antagonist (≈0% for flutamide); max = luciferase induction level with the

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positive control (≈100%); a1 = IC50 (half-maximum inhibitory concentration) and a2 = curve slope.

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When min could not be achieved because of cytotoxicity (nonachlors, PCB180 and -187) or high

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concentration not available (HCB), the curves were fitted with min set as 0. The activities of the samples ACS Paragon Plus Environment


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were calculated as flutamide-equivalents (CALUX-FluEQs/g-lipid). Concentration levels that showed

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20%–40% DHT-induced luciferase activity-inhibition were interpolated from the flutamide standard

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fitted dose–response curve. The inhibition levels (%) were also limited to ≥ three times of SD × 3 (%)

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for the EC50 level DHT in triplicate wells. All measurements were conducted in triplicate wells and

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repeated at least thrice for each concentration. Cell viability was confirmed using a

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which was described

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elsewhere.

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was considered as cytotoxic and excluded from all calculation. Receptor-mediated specificity for AR

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antagonistic activity was confirmed if the activity was mitigated by co-exposure to a high DHT dose

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(100 × EC50 level, 10 nM). 21,25

21,25

If the cell viability was -138 > -99

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> -149, and the REPs for the other congeners were less than 0.1. The α- and β-HCHs had potencies

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greater than 0.1; however, androgenic activity inhibition by β-HCH was partial. CHLs also exerted AR

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antagonism such that cis-chlordane > trans-chlordane > oxychlordane > cis-nonachlor > trans-nonachlor,

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but only the REP values for cis- and trans-chlordane were close to or greater than 0.1. Very low potency

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was observed with HCB.

24

and our previous study.

21

All the tested organochlorine compounds, except mirex,

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These results are consistent with the results of previous studies in that p,p′-DDE was the most

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potent among the DDTs and that DDTs were more potent than the chlordanes within the same cell line ACS Paragon Plus Environment


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21-23

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specific response and sensitivity of the various cell lines. HCB, nonachlors, and oxychlordane caused

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AR antagonism with U2OS cells but not with prostate cells.

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β-HCHs were higher than the γ-HCH, whereas the REPs for β- and γ-HCHs were either absent or not

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quantifiable with the other cell types.

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and -153 (0.53, 0.31, 0.74, 2.5, and 2.8 µM, respectively) in the present study was similar to the IC50

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PCB order in a previous study, which used the same assay

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-153 were also similar to the human AR-transfected CHO cells

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observed in PCB180 and -153, possibly due to inferior sensitivity. PCB99 and -187 were also reported to

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have AR antagonistic activities when human prostate cancer cells were transfected with AR (IC50 = 1.3

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and 2.1 µM, respectively).

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-183.

and with human AR-transfected CHO cells.

31,32

28,30

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However, there were differences associated with the

28,30

In this study, the REPS for α- and

The relative order of the IC50 for PCB118, -128, -138, -180,

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and the order of PCB118, -138, -180, and 17,29

results where no activity was

The present study is the first to report AR antagonism for PCB149 and

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AR Antagonistic Activities in Liver Extracts of Wild Animals

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AR-specific antagonistic activities were detected in all the striped dolphins, Stejneger’s beaked

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whales, and Steller’s sea eagles sulfuric-acid-treated liver extracts with means values and SD of 38 (20–

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52), 47 (21–96) and 80 µg CALUX-FluEQ/g-lipid, respectively (Fig. 4, Table S3). These values were

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similar to the Baikal seal livers (n = 16) FluEQs 62 µg/g-lipid

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were detected in the golden eagle liver extract (n = 1) and several extracts from finless porpoises (n =

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3/5), raccoon dogs (n = 3/5), and common cormorants (n = 5/6) with means values and ranges of 5.0, 4.4

21

in our previous study. Lower activities


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± 2.8, 12 ± 9, and 21 ± 11 µg CALUX-FluEQ/g-lipid, respectively. In the previous study, AR

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antagonistic activities were not detected in the finless porpoises, raccoon dogs or in the common

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cormorants because small extract amounts were used for exposure. 21

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Contribution of Chemicals to AR Antagonistic Activities

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The target OCPs and PCBs theoretical AR antagonistic activities on the sulfuric-acid-treated liver

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extracts were calculated according to CA model, which used the OCPs and PCBs concentrations and the

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IC25-based REP values. The theoretical values were compared with the experimental values

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(CALUX-FluEQs) (Figs. 4 and S1, Table S3) and the ratios between the total Theo-FluEQs and

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CALUX-FluEQs were 126% (67%–204%), 84%, 53% (21%–132%), 55% (17%–125%), and 44%, for

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striped dolphin, Steller’s sea eagle, Stejneger’s beaked whale, finless porpoise and golden eagle,

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respectively. The target OCPs and PCBs explained only a small portion of the CALUX-FluEQs for the

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two terrestrial species, the common cormorant [17% (10%–39%)] and raccoon dog [7.6% (1.0–48%)].

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The main AR antagonist contributor for all the samples except raccoon dog was p,p′-DDE (12%–105%

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of CALUX-FluEQs). Secondary contributors for the striped dolphin were PCB128, -118, -138 (14%

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combined), and p,p′-DDD (2.2%), whereas the secondary contributors for the finless porpoise were

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PCB138, -153, -99, -118, -128, -187 (14% combined), and p,p′-DDD (1.8%). p,p′-DDD (3.8%) and

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β-HCH (4.4%) were secondary contributors for Stejneger’s beaked whale and for the golden eagle,

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respectively. In the common cormorant, the main contributor was p,p′-DDE (12%), and the other

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components that contributed to the CALUX-FluEQs were PCB118, -128, -138, and -99 (4.1% ACS Paragon Plus Environment


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combined). Oxychlordane was the main contributor (4.5% of CALUX-FluEQs) in the raccoon dog,

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followed by PCBs (2.4% assuming that all PCB congeners were as potent as PCB128) and p,p′-DDE

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(0.27%).

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The component ratio effects on the difference between Theo-FluEQs and CALUX-FluEQs were

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explained by comparing the artificial component mixtures response curves derived from the CA and IA

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models.

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representing the 3 DDTs, 5 CHLs, and 9 PCBs mixture were compared with the respective CA-based

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Theo-FluEQs in representative liver extracts of the striped dolphins and finless porpoises, two species

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with different PCBs/DDE ratios (1/4 and 2/3, respectively). The measured effects of the artificial

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mixtures for the striped dolphins were twofold lower than the Theo-FluEQs and the observed response

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curves for the artificial mixtures were similar to response curves derived from the IA model (Fig. 5). In

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this model, it is thought that chemical constituents act upon different targets in a cell for AR antagonistic

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effect. The measured activities of the artificial mixtures for the finless porpoise samples were similar to

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the CA-based Theo-FluEQs. These findings suggest that IA and CA could be the main modes of action

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for component ratio mixtures that are similar to the ratios that are in striped dolphins and finless

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porpoises, respectively. The mechanism that causes the mode of action difference in the ratio between

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ligands is unclear. Furthermore, CALUX-FluEQs were considerably lower than DDE-FluEQs as well as

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the total Theo-FluEQs in two striped dolphins (Table S3), which could be due to an unknown occurrence

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of unidentified AR agonists in the striped dolphin livers.

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34,35

As shown in Fig. 5, the experimental AR antagonistic activities of the standard solutions

The contribution of the target OCPs and PCBs to CALUX-FluEQs was lower for the two terrestrial ACS Paragon Plus Environment

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species, the raccoon dog and common cormorant (Fig. 4, Table S3). Proximity to anthropogenic sources

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may have caused these terrestrial animals to be exposed to a greater number of chemicals; however, the

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total accumulation levels were not expected to be as high as the levels in marine species. Other persistent

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compounds may contribute to the antiandrogenic effects in these terrestrial animals. An AR antagonist,

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such as PCB170, was reported by Hamers et al.

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common cormorant (mean Theo-FluEQ = 0.066 µg/g-lipid and 0.31% of CALUX-TEQ) at the same

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level as PCB138 (0.71%) and -99 (0.62%) if the contribution was calculated with an IC50-REP (0.15)

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obtained from the IC50 PCB170 and flutamide values in the Hamers’ study and in the present study,

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respectively. PBDEs such as BDE47 and -100 are commonly found in raccoon dogs and common

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cormorants [3.2 (0.17–5.0) 10 and 51 (1.9–140) ng/g-lipid were combined for each species, respectively].

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These PBDEs (BDE47 and -100) were also known as AR antagonists

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potencies were measured in this study (IC25-REPs = 0.20 and 0.58, respectively), but the activities were

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at least one order lower than those of PCBs (data not shown). Other plausible contributors for the

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unexplained activities in these terrestrial species are unintentional analogues and transformation

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products of OCPs such as chlorinated termiticides as well as brominated and mixed halogenenated

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compounds derived from flame retardants and combustion products.10,41,43 It is important to explore and

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identify other potential unknown contributors to AR antagonistic activities in these species. The

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important finding in this study is that marine cetaceans and raptor in Japan exhibited high AR

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antagonistic activities mainly with the contribution of p,p’-DDE, while the contributors were almost

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unknown for terrestrial animals (raccoon dog and common cormorant).

23

to contribute to the AR antagonistic activities in the


25,42

and their AR antagonistic


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Toxicological risk implications for antiandrogenic chemical accumulations

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TIE approach to the acid-treated liver extracts of the seven high-trophic level animals revealed the

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effects of AR antagonistic activities (CALUX-FluEQs) and the contribution of organochlorine

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compounds for each species (Theo-FluEQs) (Figs. 4 and S1, Table S3). TIE is an important approach in

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examining the contribution of each compound, but this approach may overestimate the actual

345

contribution of each organochlorine compound when various chemicals that are in the liver extracts

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effect the different target sites in a cell, which is the case of AR antagonists in striped dolphin that is the

347

basis of IA and not CA (Fig. 5).

348

The co-existence of the target and unknown compounds may also influence the contribution of each

349

compound and a decrease of the total activities is possibly caused by unknown persistent AR agonists.

40,41

Only IA model underestimates FluEQs for real CALUX-FluEQs.

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There were few epidemiological studies about the relationship between accumulated chemicals and

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the reproductive effects for male wild animals. 3,4 However, in this study, the blood testosterone levels in

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male Dall’s porpoises were negatively correlated with p,p′-DDE and PCBs concentrations, which

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showed a significant decline of testosterone levels at more than 15 µg/g wet weight of p,p′-DDE (10 µg

354

Theo-FluEQ/g-lipid) and 10 µg/g wet weight of PCBs in blubber (7 µg Theo-FluEQ/g-lipid based on

355

REP of PCB128), 4 presuming that blubber contained mostly lipids. Previous studies suggested that male

356

gull embryos were feminized by DDTs at 20 µg/g egg weight (200 µg Theo-FluEQ/g-lipid, assuming

357

eggs contained 10% lipid) 6 and that a decline in human male reproductive health such as cryptorchidism,

358

hypospadias, and low/decreasing semen quality may have resulted from anthropogenic chemical ACS Paragon Plus Environment

Page 19 of 39


1,13,15,44

359

exposure during the fetus’ life cycle.

360

between PCB138 and sperm motility and morphology (significantly decreased at ≥0.025 µg/g-lipid in

361

serum or ≥0.0040 µg Theo-FluEQ/g-lipid) as well as limited evidence of negative correlations between

362

p,p′-DDE and sperm motility (declining trend at ≥0.30 µg/g-lipid in serum or ≥0.26 µg

363

Theo-FluEQ/g-lipid in serum).

364

inhibition on the male reproductive system may be related to the AR. In the present study, the striped

365

dolphin, Stejneger’s beaked whale, and Steller’s sea eagle had high CALUX-FluEQs (23–80 µg/g-lipid),

366

which were lower than the levels that caused feminization of the male gull embryos but higher than the

367

levels that caused the decrease of testosterone in the cetaceans serum and the levels affecting the sperm

368

quality in humans, presuming that there is not a drastic difference in the lipid-based chemical

369

concentrations in the different tissues. Thus, it is possible that species with high CALUX-FluEQ levels

370

are at risk of AR-mediated adverse effects on the development and functions of the male reproductive

371

system. It is essential to perform further detailed studies about epidemiological investigation and

372

reproductive effects on wildlife.

373

45

For instance, there was evidence of an inverse relationship

AR antagonism of these chemicals suggests that these effects of

In humans and animals, low-dose and nonmonotonic dose-response curves of chemicals may have 46

374

unclear mechanisms for chemicals that are associated with AR antagonism.

375

effects on the immunological system and the connection with the chemical pollutants (DDTs, PCBs, and

376

PBDEs) have been investigated in cetaceans and avian. 2,5,13,47,48 Approximately 100 thousand chemicals

377

have been produced and sold for industry these days and it is estimated that several thousand artificial

378

chemicals have been released into the environment.

49

Moreover, the adverse

Previous studies suggested that persistent



2,11-13,50

metabolites and non-persistent compounds

2,7,13,33,51

Page 20 of 39

379

compounds,

380

although only a few studies have used TIE for non-persistent compounds. 7 Thus, the TIE approach,

381

which uses bioassay systems that correspond to various receptors and signaling activities,

382

necessary to evaluate the diverse toxicological risks of chemicals that exert toxic effects.

383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 ACS Paragon Plus Environment

exert toxic effects on wildlife

21,23,25,52

is

Page 21 of 39

399


ACKNOWLEDGEMENTS

400

Samples were collected with the cooperation of Echizen Matsushima Aquarium, Fishery Section,

401

Otaru City, Komatsu City Museum, Komatsu City Office, Kujukushima Aquarium Umikirara, Kushu

402

University, Nagasaki Prefecture Natural Environment Division, Notojima Aquarium, Noto Marine

403

Center, Japan Cetology Study Group, Akiko Sudo (Eaglet Office), Akira Takemura (Nagasaki

404

University), Daisuke Ueno (Saga University), Haruhiko Nakata (Kumamoto University), Hiroko Koike

405

(Kyushu University), Hitoshi Hurusawa (Sapporo City Museum Activity Center), Miki Mizushima

406

(Historical Museum of Hokkaido), Osamu Sano (Ishikawa Prefectural Museum of Natural History),

407

Takahito Yamamoto (Friendship Village of Ishizuchi), and Toshio Tsubota (Hokkaido University). This

408

study was supported by Grants-in-Aid of Global Center of Excellence (GCOE) Program from the

409

Ministry of Education, Culture, Sports, Science and Technology, Japan, and Scientific Research S

410

(20221003) from the Japan Society for the Promotion of Science (JSPS). We also thank Enago

411

(www.enago.jp) for the English language review.

412 413 414 415 416 417 418 ACS Paragon Plus Environment


419

Page 22 of 39

Suppurting Information Available

420 421

Information on the tested specimens (Table S1), concentrations of individual PCBs and OCPs in

422

liver extracts (Table S2), experimental and theoretical AR antagonistic activities in liver extracts (Table

423

S3, Figure S1). This information is available free of charge via the Internet at http://pubs.acs.org.

424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 ACS Paragon Plus Environment

Page 23 of 39


439

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599 600


Table 1. AR antagonistic activities and flutamide-relative potency (REP) values (mean ± SD, n = 3 unless otherwise specified) for OCPs and PCBs. AR antagonistic activity (µM)

Standard Flutamide b OCPs p ,p '-DDT p ,p '-DDD p ,p '-DDE trans -Chlordane cis -Chlordane trans -Nonachlor cis -Nonachlor Oxychlordane α-HCH β-HCH γ-HCH HCB e Mire x PCBs PCB99 PCB118 PCB128 PCB138 PCB149 PCB153 PCB180 PCB183 PCB187

601

Cytotoxicity (µM) a

AR antagonistic REP (we ight base)

IC25

IC50

IC75

IC25

IC50

IC75

0.091 ± 0.021

0.23 ± 0.061

0.60 ± 0.24

1

1

1

0.39 ± 0.036 0.28 ± 0.037 0.095 ± 0.017 0.78 ± 0.079 0.36 ± 0.066 4.3 ± 1.2 2.2 ± 0.36 0.97 ± 0.090 0.49 ± 0.13 0.93 ± 0.038 1.5 ± 0.24 1.6 n.d. f

1.0 ± 0.0098 0.69 ± 0.14 0.25 ± 0.035 1.7 ± 0.068 1.0 ± 0.10 7.9 ± 2.0 – 2.7 ± 0.21 1.3 ± 0.33 – 4.0 ± 0.19 – n.d.

2.7 ± 0.21 2.1 ± 0.61 0.65 ± 0.12 3.6 ± 0.61 2.8 ± 0.78 – d – 7.6 ± 0.67 3.5 ± 0.83 – 10 ± 1.8 – n.d.

0.19 ± 0.010 0.29 ± 0.029 0.87 ± 0.046 0.076 ± 0.0095 0.19 ± 0.055 0.015 ± 0.0031 0.024 ± 0.0044 0.053 ± 0.0085 0.15 ± 0 0.11 ± 0.023 0.057 ± 0.0095 0.059 n.d.

0.20 ± 0.015 0.26 ± 0.010 0.78 ± 0.026 0.096 ± 0.017 0.15 ± 0.020 0.022 ± 0.0036 – 0.048 ± 0.0085 0.15 ± 0.012 – 0.065 ± 0.0067 – n.d.

0.21 ± 0.042 0.24 ± 0.036 0.70 ± 0.040 0.12 ± 0.031 0.12 ± 0.027 – – 0.045 ± 0.014 0.15 ± 0.017 – 0.078 ± 0.021 – n.d.

30 30 ND c 30 30 30 10 ND ND ND ND ND ND

1.3 0.53 0.31 0.74 1.7 2.8 2.5 2.6 2.1

3.2 1.2 0.87 1.4 4.4 6.3 4.0 6.4 4.6

0.16 ± 0.010 0.31 ± 0.056 0.73 ± 0.16 0.16 ± 0.015 0.12 ± 0.021 0.047 ± 0.013 0.038 ± 0.0060 0.060 ± 0.0098 0.073 ± 0.0080

0.14 ± 0.0058 0.31 ± 0.036 0.71 ± 0.11 0.21 ± 0.017 0.11 ± 0.025 0.052 ± 0.012 0.057 ± 0.010 0.056 ± 0.011 0.074 ± 0.0068

0.13 ± 0.015 0.31 ± 0.026 0.71 ± 0.21 0.27 ± 0.030 0.11 ± 0.028 0.058 ± 0.014 0.089 ± 0.026 0.052 ± 0.013 0.073 ± 0.0064

30 ND 30 30 30 30 30 30 30

0.54 0.24 0.12 0.41 0.69 1.3 1.6 1.1 0.97

± 0.096 ± 0.057 ± 0.035 ± 0.12 ± 0.21 ± 0.32 ± 0.26 ± 0.27 ± 0.14

± 0.19 ± 0.044 ± 0.031 ± 0.071 ± 0.48 ± 0.26 ± 0.11 ± 0.48 ± 0.19

± 0.38 ± 0.11 ± 0.16 ± 0.37 ± 1.3 ± 0.70 ± 0.32 ± 1.0 ± 0.32

a . the concentration for which the cell viability

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