Peroxisome Proliferator-Activated Receptor γ is a Sensitive Target for

Publication Date (Web): June 24, 2016 .... Fresh OPSW was collected from the West-In-Pit tailings pond (Syncrude Canada Ltd., Fort McMurray, AB, Canad...
0 downloads 0 Views 1MB Size
Subscriber access provided by Purdue University Libraries

Article

Peroxisome Proliferator-Activated Receptor # is a Sensitive Target for Oil Sands Process-affected Water: Effects on Adipogenesis and Identification of Ligands Hui Peng, Jianxian Sun, Hattan A. Alharbi, Paul D. Jones, John P. Giesy, and Steve B. Wiseman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01890 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016

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 27

Environmental Science & Technology

1

Peroxisome Proliferator-Activated Receptor γ is a Sensitive Target for Oil Sands

2

Process-affected Water: Effects on Adipogenesis and Identification of Ligands

3 4

Hui Penga, Jianxian Sun*a, Hattan A. Alharbia, Paul D. Jonesa,b, John. P. Giesy*a, c,d,e,f and Steve Wisemana,g

5 6 a

7 8 9 10 11 12 13 14 15 16 17 18 19 20

Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, Canada, S7N 5B3 b School of Environment and Sustainability, 117 Science Place, Saskatoon, SK, Canada, S7N 5C8 c Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B3 d Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA e State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China f Biology Department, Hong Kong Baptist University, Hong Kong, SAR, China g Department of Biology, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.

21

*Corresponding authors: Jianxian Sun, e-mail: [email protected]; John. P. Giesy,

22

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N5B3, Canada;

23

TEL (direct): 306-966-2096; TEL (secretary): 306-966-4680; FAX: 306-966-4796; e-mail:

24

[email protected]

1

ACS Paragon Plus Environment

Environmental Science & Technology

25

ABSTRACT

26

Identification of toxic components of complex mixtures is a challenge. Here, oil sands

27

process-affected water (OSPW) was used as a case study to identify those toxic components

28

with a known protein target. Organic chemicals in OSPW exhibit dose-dependent activation

29

of peroxisome proliferator-activated receptor γ (PPARγ) at concentrations less than those

30

currently in the environment (0.025× equivalent of full-strength OSPW), by use of a

31

luciferase reporter gene assay. Activation of PPARγ-mediated adipogenesis by OSPW was

32

confirmed in 3T3L1 preadipocytes, as evidenced by accumulation of lipids and up-regulation

33

of AP2, LPL and PPARγ gene expression after exposure to polar fractions of OSPW.

34

Unexpectedly, the nonpolar fractions of OSPW inhibited differentiation of preadipocytes via

35

activation of the Wnt signaling pathway. Organic chemicals in OSPW that were ligands of

36

PPARγ were identified by use of a pull-down system combined with untargeted chemical

37

analysis (PUCA), with a recombinant PPARγ protein. Thirty ligands of PPARγ were identified

38

by use of the PUCA assay. High resolution MS1 and MS2 spectra were combined to predict

39

the formulas or structures of a subset of ligands, and polyoxygenated or heteroatomic

40

chemicals, especially hydroxylated carboxylic/sulfonic acids, were the major ligands of

41

PPARγ.

42 43 44 45

KEYWORDS: OSPW; Untargeted chemical analysis; Pull down; His-tagged recombinant

46

protein; Orbitrap ultrahigh resolution mass spectrometry.

47 2

ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27

48

Environmental Science & Technology

INTRODUCTION

49

In the surface mining oil sands industry in Northern Alberta, Canada, extraction of

50

bitumen from oil sands results in production of oil sands process-affected water (OSPW).

51

Rather than releasing OSPW back to the receiving environment, OSPW is stored in tailings

52

ponds and settling basins, and recycled for extraction of bitumen.

53

area of approximately 170 km2 and contain greater than 1 billion m3 of OSPW.1 After a

54

surface mine is closed or when OSPW is no longer useful for extraction of bitumen, tailings

55

ponds must be reclaimed and detoxified, but as yet, no method to achieve this at the scale

56

required in this industry has been developed.2

57

major strategy for reclamation and detoxification of OSPW.

58

constructed in mined-out pits of oil sands mines, will be permanent features of reclaimed

59

landscapes, and will be hydraulically connected with the natural environment.3

60

30 EPLs have been planned by various companies operating in the surface mining industry,4

61

so demonstrating success of this strategy is important. However, because there is concern

62

with regard to the rate at which OSPW in EPLs will be detoxified, effectiveness of EPLs for

63

eliminating toxicity of OSPW was called into question by a report by the Royal Society of

64

Canada.4

65

assist industry and regulatory agencies charged with monitoring EPLs.

66

include bioassays that allow for monitoring of toxicity or high resolution mass spectrometry

67

assays to monitor concentrations of specific chemicals that cause adverse effects.

Tailings ponds cover an

End-pit lakes (EPLs) have been proposed as a In general, EPLs will be

Greater than

Development of tools to monitor detoxification of OSPW in EPLs would greatly These tools could

68

Implementation of bioassays or mass spectrometry assays to monitor detoxification of

69

EPLs is dependent on a comprehensive knowledge of adverse effects caused by exposure to

3

ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 27

70

OSPW and identification of specific chemicals that cause these effects.

71

based on the critical mechanism of toxicity, or that adverse effect that occurs at the least

72

concentration of chemicals.

73

impaired growth and development,6 endocrine disruption and impairment of reproduction,7, 8

74

and immunotoxicity,

75

effects.

76

complexity of OSPW. Analysis of OSPW by use of ultrahigh-resolution mass spectrometry

77

has revealed thousands of chemicals, including species containing oxygen, nitrogen, and

78

sulfur.11-13

79

that cause acute lethality5, 14, 15 and endocrine disruption.16, 17 However, the EDA strategy has

80

some limitations.

81

mixture are present in “active” fractions, and each chemical in an “active” fraction might not

82

contribute to biological effects.

83

fractionation of organic chemicals in the aqueous phase of OSPW, two fractions that caused

84

acute lethality were produced, but several classes of chemicals were present in each fraction,

85

and it is not known if each of the classes of chemicals was responsible for acute lethality.

86

Alternatively, when the mode of toxic action involves binding of chemicals to a protein, it is

87

possible to use the protein target as an affinity matrix to specifically capture bioactive

88

metabolites or drugs.18 Using this strategy, physically-interacted ligands can be identified with

89

a lesser false positive rate, and laborious fractionation of samples is not necessary. However,

90

because the high background interferences produced from the process decreases sensitivity

91

and specificity of the method, application of the strategy to environmental matrices is a great

9, 10

Bioassays should be

Adverse effects of exposure to OSPW include acute lethality,5

but little is known about specific toxic components that cause these

One major impediment to identification of toxic components is the chemical

Effects directed analysis (EDA) has been used to identify chemicals in OSPW

For example, it is not known if all the causative chemicals in a complex

In the study by Morandi et al,14 after three rounds of

4

ACS Paragon Plus Environment

Page 5 of 27

Environmental Science & Technology

92

challenge because of the complexity and low abundance of ligands in environmental samples.

93

Therefore, it is necessary to develop a more robust strategy to improve identification of toxic

94

components in OSPW that cause adverse effects.

95

Naphthenic acids (NAs), which are a major chemical constituent of OSPW, are a group of

96

cyclic and acyclic, alkyl-substituted carboxylic acids with the general formula of CnH2n+ZO2.19

97

It has been suggested that some NAs are structurally similar to fatty acids.19, 20

98

acids are ligands of peroxisome proliferator-activated receptor γ (PPARγ),21,

99

receptor that is a ligand activated transcription factor and important regulator of adipogenesis,

Because fatty 22

a nuclear

100

PPARγ might be an important target of OSPW.

101

contaminants, such as tributyltin (TBT) and triphenyl phosphate (TPP), causes adverse effects

102

such as promotion of adipogenesis resulting in weight gain and obesity.23-26 Therefore,

103

disruption of PPARγ signaling might be an important mechanism of toxicity of OSPW.

104

Activation of PPARγ by environmental

The goal of the current study was to test the hypothesis that ligands of PPARγ are present

105

in OSPW and to identify these ligands.

Activation of PPARγ signaling was determined by

106

use of a reporter assay and promotion of adipogenesis was evaluated by use of 3T3L1 cells.

107

Finally, a novel method to identify ligands of PPARγ was developed in which a pull-down

108

assay with recombinant PPARγ protein was combined with untargeted chemical analysis

109

(PUCA) by use of Orbitrap ultrahigh-resolution mass spectrometry.

110 111

Materials and Methods

112

Chemicals and Reagents. Details are provided in Supporting Information.

113

OSPW Sample Collection and Extraction. Samples of OSPW from two sources were

5

ACS Paragon Plus Environment

Environmental Science & Technology

114

investigated in the present study. Fresh OPSW was collected from the West-In-Pit tailings

115

pond (Syncrude Canada Ltd., Fort McMurray, AB, Canada). The West-in-Pit settling basin

116

was commissioned as Base Mine Lake (BML-OSPW), which is the first end pit lake in the oil

117

sands industry, in December 2012. A sample of aged OSPW also was collected from an

118

experimental reclamation pond called Pond 9 (P9- OSPW) that was constructed in 1993 and

119

has not received input of OSPW since that time. All samples were collected in September of

120

2012, shipped to the University of Saskatchewan (Saskatoon, SK, Canada), and stored in the

121

dark, and used for fractionation immediately upon arrival.14, 27 Fractionation of BML-OSPW

122

samples (n=3) or P9-OSPW (n=1) was conducted by use of EVO-LUTE® ABN SPE

123

cartridges (Biotage, Charlotte, NC, USA) because of their ability to extract a broad range of

124

chemicals, as demonstrated in our previous study.27 Preliminary data also showed that PPARγ

125

activity of BML-OSPW extracted by EVO-LUTE® ABN SPE cartridges was greater than

126

activity of the same sample of OSPW extracted by HLB cartridges. A procedure blank was

127

conducted using ultrapure water, and no significant activity of PPARγ was detected. All the

128

extracts were dissolved in ethanol and stored at -20 oC. Details of the sample pretreatment are

129

provided in Supporting Information.

130

PPARγ Assay. Activation of PPARγ was determined by use of a human PPARγ reporter assay,

131

according to the protocol provided by the manufacturer (Cayman Chemical, Ann Arbor, MI,

132

USA), which is described in Supporting Information. Antagonism of PPARγ was determined

133

by mixing 50 nM of rosiglitazone (~EC50), which is an agonist of PPARγ, with samples of

134

BML-OSPW or T0070907 (antagonist of PPARγ). Antagonistic activity was determined

135

using the same method used to determine activation of PPARγ. All exposures were conducted

6

ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27

Environmental Science & Technology

136

in triplicate (n=3).

137

Differentiation of 3T3-L1 Cells. Adipogenesis was assessed using 3T3L1 preadipocytes

138

(ATCC® CL-173™, Manassas, VA) as described previously28 and in the Supporting

139

Information. Expression of genes that are regulated by PPARγ (PPARγ, AP2, LPL and WISP2)

140

were quantified by use of quantitative real-time PCR (qPCR). Expression of target genes was

141

normalized to expression of 36B4.28, 29 Details of the qPCR protocol are provided in the

142

Supporting Information.

143

His-PPARγ Pull-Down Assay. Details of the pull-down experiments to identify ligands of

144

PPARγ are described in Supporting Information.

145

Mass Spectrometry and Untargeted Data Processing. Aliquots of extracts were analyzed

146

using a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Dionex™

147

UltiMate 3000 UHPLC system.

148

accomplished with an in-house R program as described in the Supporting Information.

Analysis of untargeted mass spectrometry data was

149 150

Results and Discussion

151

PPARγ Agonistic Activity of OSPW. The total extract (TE) of organic chemicals from the

152

aqueous phase of BML-OSPW significantly activated PPARγ-driven reporter activity and the

153

effect was dose-dependent (Figure 1A). Maximum activation was 14.6±0.19-fold (relative to

154

vehicle control; corresponding to 21.8% of the maximal activity of rosiglitazone at 625 nM)

155

in response to a 1× equivalent of the TE.

156

activity (1.3±0.12-fold, p=0.037) was observed at concentrations as small as 0.025×, which

157

represents a 40-fold dilution of dissolved organic chemicals in BML-OSPW.

158

bioassay-derived rosiglitazone equivalent (REQ) of BML-OSPW was calculated to be 55.7

159

nM (Figure S1A, Supporting Information). Dose-dependent activation of PPARγ also was

Significant activation of PPARγ-driven reporter

7

ACS Paragon Plus Environment

The

Environmental Science & Technology

Page 8 of 27

160

detected in two other replicate samples of BML-OSPW (Figure S1B). Previous studies have

161

reported nuclear receptor activities or other toxicities of OSPW, but no other study has

162

reported an effect of OSPW at such a small concentration (40× dilution).

163

the EPL strategy, OSPW will be diluted by input of waters from natural surface and

164

groundwater, runoff and precipitation.3

165

ensure that dilution of OSPW is adequate to ameliorate effects on PPARγ.

166

of OSPW from tailings ponds to surface water has been reported,30 it would be appropriate to

167

determine if these surface waters have greater potential to activate PPARγ than natural surface

168

waters not impacted by seepage from tailings ponds. Activation of PPARγ by P9-OSPW was

169

less compared to activation by BML-OSPW (Figure S1B), and the response at 1× was only

170

5.9±0.4% of the maximal activity of rosiglitazone at 625 nM. These results suggest that aging

171

of OSPW is effective to detoxify OSPW.

7, 14, 16

According to

These results suggest that it will be important to Because seepage

172

To further investigate the PPARγ activity of BML-OSPW, the replicate causing greatest

173

activation (replicate-1) was selected for further fractionation by use of SPE cartridges.

174

Consistent with effects of the TE, each of the five fractions of BML-OSPW activated

175

PPARγ-driven reporter activity (Figure 1B).

176

DCM in hexane as the elution solvent) and F5 (methanol as the elution solvent), with

177

induction of 9.6±0.55-fold (p