High-throughput in vitro Data To Inform Prioritization of Ambient Water

Dec 7, 2017 - There is currently no systematic or national prioritization for monitoring waters for chemicals with endocrine disrupting activity. We p...
2 downloads 11 Views 1MB Size
Subscriber access provided by READING UNIV

Article

High-throughput in vitro Data To Inform Prioritization of Ambient Water Monitoring and Testing for Endocrine Active Chemicals Wendy J. Heiger-Bernays, Susanna Wegner, and David Dix Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00014 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 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 31

1 2 3 4 5 6 7 8 9

Environmental Science & Technology

High-throughput in vitro Data To Inform Prioritization of Ambient Water Monitoring and Testing for Endocrine Active Chemicals a

Wendy J. Heiger-Bernaysa*, Susanna Wegnerb and David Dixc Boston University School of Public Health, 715 Albany St. Boston, MA 02118, USA b Oak Ridge Institute of Science and Education, Oak Ridge, TN, USA c DJD Consulting, PO Box 4518, Paso Robles, CA 93447, USA

*Department of Environmental Health, (617) 358-2431; [email protected]

ACS Paragon Plus Environment

1

Environmental Science & Technology

10

ABSTRACT

11

The presence of industrial chemicals, consumer product chemicals, and pharmaceuticals is well

12

documented in waters in the US and globally. Most of these chemicals lack health-protective

13

guidelines and many have been shown to have endocrine bioactivity. There is currently no

14

systematic or national prioritization for monitoring waters for chemicals with endocrine

15

disrupting activity. We propose Ambient Water Bioactivity Concentrations (AWBCs) generated

16

from high throughput data as a health-based screen for endocrine bioactivity of chemicals in

17

water. The US EPA ToxCast program has screened over 1800 chemicals for estrogen receptor

18

(ER) and androgen receptor (AR) pathway bioactivity. AWBCs are were calculated for 110 ER

19

and 212 AR bioactive chemicals using high throughput ToxCast data from in vitro screening

20

assays and predictive pathway models, high-throughput toxicokinetic data, and data-driven

21

assumptions about consumption of water. Chemical-specific AWBCs are compared with

22

measured water concentrations in datasets from the greater Denver area, Minnesota lakes, and

23

Oregon waters, demonstrating a framework for identifying endocrine bioactive chemicals. This

24

approach can be used to screen potential cumulative endocrine activity in drinking water and to

25

inform prioritization of future monitoring, chemical testing and pollution prevention efforts.

Page 2 of 31

26 27

INTRODUCTION

28

Since the 1990s, contaminants of emerging concern (CECs) have been measured in US surface

29

waters,1,2 groundwaters3 and drinking water4,5 and waters globally.6,7 CECs include agents that

30

are unregulated in water supplies or those which lack sufficient toxicological data for decision-

31

making. Unregulated CECs such as pharmaceuticals, personal care products, and agricultural2 or

32

industrial2 chemicals enter into water supplies from municipal wastewater treatment plants8,9

ACS Paragon Plus Environment

2

Page 3 of 31

Environmental Science & Technology

33

croplands,10 industrial and commercial facilities,11 septic systems,12 landfills13 and concentrated

34

animal feeding operations.14 The growing need for recycled water coupled with increasing

35

drought conditions15 heighten the need for new tools to aid in assessment of potential human and

36

ecological risks associated with CECs in water. Many of the CECs detected in water are of

37

particular concern due to their potential to alter hormone signaling pathways in living systems at

38

relatively low concentrations.16 The biological signaling pathways that regulate brain

39

development, reproductive capabilities, growth and behavior are exquisitely sensitive to very

40

small changes in concentration and temporal presence of chemicals with endocrine activity,16

41

including endocrine signaling molecules. While there are some state and regional water

42

monitoring efforts, the US lacks a systematic national monitoring program that prioritizes CECs

43

that have endocrine bioactivity.

44

The U.S. EPA’s Endocrine Disrupting Screening Program (EDSP) is responsible for evaluating

45

potential endocrine effects of all pesticide active and inert ingredients, and chemicals found in

46

drinking water sources with the potential for significant human exposures that conceivably could

47

include many chemicals in commerce.17 In the 20 years since the EDSP legislative mandate, only

48

52 pesticides have been evaluated in the current screening battery of tests, and no chemicals have

49

been conclusively designated by U.S. EPA for regulation as endocrine disruptors. However,

50

there are an estimated 10,000 unique chemicals in the EDSP universe of chemicals that are

51

candidates for testing for potential endocrine activity.18 Understanding the prevalence and

52

bioactivity of these chemicals in current and potential drinking water sources is only now

53

possible with newly validated high throughput tools developed in the EDSP to identify chemicals

54

with potential endocrine activity.19

ACS Paragon Plus Environment

3

Environmental Science & Technology

Page 4 of 31

55

In the past five years, the U.S. EPA has made an effort to replace existing testing requirements

56

for endocrine activity with high throughput in vitro screening assays. The most progress has been

57

made on the estrogen receptor and androgen receptor pathways. U.S. EPA's Toxicity Forecaster

58

(ToxCast) program has developed a panel of high throughput in vitro screening assays and

59

computational toxicology methods to identify chemicals with estrogen receptor (ER) bioactivity

60

and/or androgen receptor (AR) bioactivity. A computational network model is used to integrate

61

in vitro assay responses for chemicals into the ER or AR pathway based on the molecular events

62

that typically occur in a steroid hormone receptor-mediated response. To identify ER agonist and

63

antagonist bioactivity,20,21 the ToxCast ER pathway model integrates concentration:response data

64

from 18 in vitro high throughput assays. To identify AR agonist and antagonist bioactivity,22 the

65

ToxCast AR pathway model integrates concentration:response data from nine in vitro high

66

throughput assays. Assays included in these pathway models target a range of molecular events,

67

including receptor binding, dimerization, co-factor recruitment, DNA binding, and protein

68

production. The output of these models provides a score of potential ER or AR agonist and

69

antagonist activity, chemical potency, and a measure of assay-specific false positive activity of

70

each chemical run in ToxCast.21,22 These models have been shown in multiple reviews and

71

analyses to perform at least as well as validated toxicological assays in identifying endocrine

72

bioactivity. 18, 21,22

73 74

The U.S. EPA develops Ambient Water Quality Criteria (AWQC) for the protection of

75

human health and the environment under the US Clean Water Act.24 AWQCs are designed to

76

protect and maintain quality of surface waters for potential use as drinking water, not only those

77

waters that are currently used for drinking water. Promulgated AWQCs are derived from

ACS Paragon Plus Environment

4

Page 5 of 31

Environmental Science & Technology

78

traditional in vivo toxicological data from which points of departure are identified. They provide

79

a point of reference for interpretation of potential risks associated with chemical concentrations

80

detected in the environment. However, AWQCs are not available for the large number of

81

contaminants frequently detected in water, presenting a challenge for interpretation of

82

monitoring data on data-poor chemicals, including potential endocrine disrupting chemicals.

83

In the absence of AWQCs, high-throughput in vitro data and computational models such

84

as EPA’s ToxCast program23 provide endocrine bioactivity data that can put monitoring data on

85

otherwise data-poor contaminants into context. To inform monitoring efforts for the large

86

number of data-poor chemicals, we propose the use of Ambient Water Bioactivity

87

Concentrations (AWBCs) derived from high-throughput ToxCast data. We present a

88

methodology to develop AWBCs as analogs to AWQCs, using an approach analogous to

89

promulgated AWQCs. An AWBC is a chemical-specific reference concentration in water that is

90

based on endocrine bioactivity of the chemical in the high-throughput assays and is protective of

91

human health. By combining AWBCs, cumulative potential for bioactivity can be assessed for

92

mixtures of chemicals identified in individual water samples. This is the first use of high

93

throughput in vitro toxicological data for deriving human health screening level concentrations

94

for comparison to measured concentrations of CECs in US waters. The aims of this manuscript

95

are: 1) to present a generalizable framework for deriving screening level human health AWBCs

96

using ToxCast data; 2) to demonstrate this approach for estrogen receptor (ER) and androgen

97

receptor (AR) bioactive chemicals; 3) to illustrate how measured water concentrations of ER and

98

AR bioactive chemicals can be compared with chemical-specific AWBCs; and 4) to extend the

99

single chemical methodology to estimating bioactivity in chemical mixtures for multiple ER and

ACS Paragon Plus Environment

5

Environmental Science & Technology

Page 6 of 31

100

AR bioactive chemicals, showing how AWBCs can be used to evaluate potential for cumulative

101

endocrine bioactivity.

102

EXPERIMENTAL (MATERIALS and METHODS)

103

Selection of ER and AR Bioactive Chemicals

104

ToxCast pathway models were developed for estrogen and androgen (ER and AR) and used to

105

produce an overall activity score for each chemical tested on a scale of 0-1.20,21,22 For this

106

analysis, chemicals with overall ER or AR bioactivity rankings equal to or greater than 0.1 are

107

considered bioactive. The ER and AR models were validated based on high balanced accuracy in

108

identification of sets of reference chemicals.20,22 Assay-specific concentration:response curves

109

modeled for each chemical were used to identify median concentrations producing 50% of

110

maximum (AC50) responses across all active assays, providing an estimate of activity in a

111

biologically relevant model.21,22 The AC50s were selected because they represent the average

112

responses across the 18 ER and nine AR assays, decreasing the influence of outlier responses

113

from individual assays. Proprietary pharmaceuticals included in ToxCast (largely candidate

114

compounds donated by pharmaceutical companies for ToxCast testing that are not currently

115

marketed) were excluded from the current analysis as they are unlikely to be present in water and

116

they cannot be identified for monitoring in US waters.

117

Conversion between In Vitro Concentrations and Exposure Rates

118

U.S EPA has developed high throughput toxicokinetic models (HTTK) that predict human doses

119

of chemicals based on the associated bioactivity in vitro concentrations. Reverse dosimetry

120

assuming first-order metabolism using toxicokinetic (TK) modeling was used to predict the oral

121

equivalent dose (OED) of a chemical needed to produce an internal (plasma) concentration equal

122

to a bioactive in vitro concentration. 25, 26, 27 Monte Carlo simulations informed by experimental

ACS Paragon Plus Environment

6

Page 7 of 31

Environmental Science & Technology

123

data on protein binding and metabolism have been used to estimate a distribution of steady state

124

blood concentrations across a hypothetical population (based on exposure assumptions for a 35

125

year old Caucasian male) with 1 mg/kg/day of exposure to a given chemical. 25, 26, 27, 28 We use

126

the population 95th percentile of these previously simulated blood concentrations as a

127

conservative set of conversion factors (to convert concentrations to doses) to estimate the oral

128

equivalent dose (OED) in mg/kg/day required to achieve blood steady state (Css) levels identical

129

to the bioactive concentrations in the ToxCast assays. For the 150 chemicals lacking

130

experimentally derived HTTK data, the geometric mean of the HTTK conversion factors

131

available for the other 95 ER and AR active chemicals were used to estimate exposure rates.

132

Calculation of the Ambient Water Bioactivity Concentrations (AWBC)

133

An Ambient Water Bioactivity Concentration (AWBC) was calculated for each ER and AR

134

bioactive chemical and its respective OED using an approach similar to that used by the US EPA

135

to develop Ambient Water Quality Criteria29 as follows:

136

Equation 1:   =  

   





 

 



!"



#  1,000,000 

137 138 139 140 141 142 143 144

where OED (mg/kg-day) = Oral Equivalent Dose = ToxCast Bioactivity(µM) x ((1 mg/kg)-day)/HTTKCss

145

vivo extrapolation already accounts for variability and sensitivity in the population. The

146

AWBCAdult is based on assumptions describing US adult population characteristics for body

147

weight and daily water consumption. The Daily Intake (DI) of 2.4 L/day and Body Weight (BW)

148

of 80 kg represent the per capita estimate of combined direct and indirect community water

(µM)

RSC = Relative Source Contribution, unitless (EPA, 2000) BW = Body Weight (kg) DI = Drinking Water Intake (liters/day) The OED is not modified by application of traditional uncertainty factors since the in vitro to in

ACS Paragon Plus Environment

7

Environmental Science & Technology

Page 8 of 31

149

ingestion at the 90th percentile for adults ages 21 and older and associated body weights30. The

150

AWBCInfant is based on assumptions describing infants for whom reconstituted infant formula

151

and water are the major sources of nutrition and liquids for the first six months of life. Daily

152

Intake (DI) and Body Weights (BW) for infants 0-1 month, 1-3 months and 3-6 months were

153

used to calculate 95th percentile population estimates for the most vulnerable population: the 0-1

154

month infant30. The Relative Source Contribution (RSC) is a factor used to account for the

155

percentage of a person’s exposure to chemicals that may come from drinking water. The EPA

156

default factor of 0.2, indicating that 80% of a person’s exposure comes from a non-drinking

157

water source, is assigned, although this factor should be modified based on exposure data

158

specific for each chemical29. The Daily Intake (DI) of 0.184 L/day and Body Weight (BW) of 4.8

159

kg represent the per capita estimate of water ingestion at the 95th percentile for infants under 3

160

months and mean body weight31. Inputs and calculated AWBCs are included in Table S-1. For

161

bioactivity-based prioritization of chemicals for monitoring, we identify priority ER and AR

162

active chemicals, those with an AWBCInfant