Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Ecotoxicology and Human Environmental Health
Genotoxicity Assessment of Drinking Water Disinfection By-products (DBPs) by DNA Damage and Repair Pathway Profiling Analysis Jiaqi Lan, Sheikh Mokhlesur Rahman, Na Gou, Tao Jiang, Michael J Plewa, Akram Alshawabkeh, and April Z Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06389 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018
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 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 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.
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
TOC
ACS Paragon Plus Environment
Environmental Science & Technology
1
Genotoxicity Assessment of Drinking Water
2
Disinfection By-products (DBPs) by DNA
3
Damage and Repair Pathway Profiling Analysis
4
5
Jiaqi Lan1,2, Sheikh Mokhlesur Rahman 1, Na Gou1, Tao Jiang1, Micheal J. Plewa3, Akram
6
Alshawabkeh1 and April Z.Gu1,4*
7
1
8
Avenue, Boston, Massachusetts 02115, US.
9
2
Department of Civil and Environmental Engineering, Northeastern University, 360 Huntington
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
10
College, Beijing, 100050, China
11
3
12
Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, US.
13
4
14
*corresponding author:
[email protected] Safe Global Water Institute, University of Illinois at Urbana-Champaign and the Department of
School of Civil and Environmental Engineering, Cornell University, Ithaca, NY, 14850, US.
15
ACS Paragon Plus Environment
Page 2 of 34
Page 3 of 34
16 17
Environmental Science & Technology
ABSTRACT Genotoxicity is considered a major concern for drinking water disinfection by-products
18
(DBPs). Of over 700 DBPs identified to date, only a small number of has been assessed with
19
limited information for DBP genotoxicity mechanism(s). In this study we evaluated genotoxicity
20
of 20 regulated and un-regulated DBPs applying a quantitative toxicogenomics approach. We
21
used GFP-fused yeast strains that examine protein expression profiling of 38 proteins indicative
22
of all known DNA damage and repair pathways. The toxicogenomics assay detected
23
genotoxicity potential of these DBPs in consistent with conventional genotoxicity assays
24
endpoints. Furthermore, the high-resolution, real-time pathway activation and protein expression
25
profiling, in combination with clustering analysis, revealed molecular level details in the
26
genotoxicity mechanisms among different DBPs and enabled classification of DBPs based on
27
their distinct DNA damage effects and repair mechanisms. Oxidative DNA damage and base
28
alkylation were confirmed to be the main molecular mechanisms of DBP genotoxicity. Initial
29
exploration of QSAR modeling using moleular genotoxicity endpoints (PELI) suggested that
30
genotoxicity of DBPs in this study was mainly contributed by topological and quantum chemical
31
descriptors. This study presents a toxicogenomics-based assay for fast and efficient mechanistic
32
genotoxicity screening and assessment of large number of DBPs. The results help to fill in the
33
knowledge gap in the understanding of the molecular mechanisms of DBP genotoxicity.
34
ACS Paragon Plus Environment
Environmental Science & Technology
35
INTRODUCTION
36
Drinking water disinfection by-products (DBPs) are formed during the reaction of disinfectants
37
(such as chlorine, chlorine dioxide, chloramine, UV and ozone) with naturally occurring organic
38
matter (NOM) and other contaminants present in water.1 DBPs are therefore widely existed in
39
drinking water at sub- µg/L (ppb) to low-to-mid- µg/L levels. Currently, there are over 700 DBPs
40
reported in drinking water, and new DBPs are continuing to be uncovered. 1-3
41
Great knowledge gaps exist for toxicological information and health impacts of DBPs.
42
Humans are exposed to DBPs through multiple routes, including ingestion (the common route
43
studied), inhalation and dermal exposures. 3 Literature review indicates that only approximately
44
15% (~100) of identified DBPs have been assessed with in vitro bioassays and a few with
45
chronic in vivo studies.2-4 Potential health risks of DBPs have been reported, including cancer
46
and other adverse reproductive effects, such as early-term miscarriage and birth defects. 5-7 An
47
association of specific cancers and exposure to disinfected water has emerged by
48
epidemiological research. 5, 8, 9 Several toxicity mechanisms for DBPs have been implicated,
49
including genotoxicity, oxidative stress, disruption of folate metabolism and cell cycle
50
disruption.5, 8, 9 Genotoxicity is of particular importance because of its link to mutagenicity,
51
carcinogenicity as well as cancer. 7, 10
52
The genotoxicity of evaluated DBPs seemed to be dependent on the structure and the
53
substituents. For example, among the halogenated DBPs, iodinated DBPs were observed to be
54
more toxic than their brominated and chlorinated analogs. 11, 12 Nitrogen-containing DBPs were
55
more genotoxic than the DBPs that do not contain nitrogen. 3, 13 Genotoxicity mechanisms of
56
DBPs are also strongly related to their structures. For example, for halogenated DBPs, oxidative
57
stress-induced DNA damage 14-21 and DNA alkylation 22-25 are two major mechanisms.
ACS Paragon Plus Environment
Page 4 of 34
Page 5 of 34
Environmental Science & Technology
58
Halonitriles may also induce genomic damage by cell cycle disruption and the induction of
59
hyperploidy. 26 Nitrosamines may act as alkylating agents after metabolism, 27 and formaldehyde
60
can form DNA–protein crosslinks. 24 The genotoxicity and mechanisms of most DBPs remain
61
unknown.
62
The standard and most reliable genotoxicity tests are in vivo assays, however, they are
63
resource-intensive and time-consuming, therefore cannot meet the demand for evaluating a large
64
number of potential genotoxic DBPs.28, 29 In vitro genotoxicity assays, including Ames test,
65
comet test and micronucleus test, require relatively shorter testing time (several days), but often
66
yield inconsistent or false results compared to in vivo outcomes 7, 30, 31 due to the inherent
67
limitations of the target and DNA damage effects they can detect.7, 30 In recent years, high-
68
throughput genotoxicity assessment has been reported where the activation of single or selected
69
biomarkers indicative of DNA damage recognition and repair are used to indicate potential
70
genotoxicity.7, 32-37 Our group has recently developed and validated a new quantitative
71
toxicogenomics-based assay based on real time protein expression profiling of known DNA
72
damage and repair pathways ensemble. 38-41 Compared to other biomarkers-based tests, our assay
73
derives quantitative endpoints that correlate with conventional genotoxicity endpoints and
74
promises to be a cost-effective and mechanistic genotoxicity assessment assay. 40, 41
75
In this study, we employed the newly developed quantitative toxicogenomics
76
genotoxicity assay to perform a mechanistic genotoxicity assessment and profiling of 20 DBPs
77
representing nine different chemical classes of DBPs. The results provided new genotoxicity
78
information and insights of underlying DNA damaging mechanisms at molecular level for these
79
20 DBPs. The high-resolution protein expression profiles of DNA damage and repair pathways
ACS Paragon Plus Environment
Environmental Science & Technology
80
also enabled DBP classification and further exploration of association between DBP chemical
81
structure and the genotoxicity mechanisms.
82 83
MATERIALS AND METHODS
84
Chemicals
85
20 DBPs were selected that belong to nine different chemical classes (Table 1,
86
manufacturer information in Table S1). Each DBP was evaluated across six-log sub-cytotoxic
87
concentration range (Table S1). The maximum non-cytotoxic concentration was pre-determined
88
(> 95% cell survival tested by growth inhibition in yeast for 24 hours, Figure S1).
89
Yeast Whole Cell Array and Real Time Protein Expression Analysis upon DBP Exposures
90
The whole cell assay library consists 38 in frame GFP fusion proteins (Table S2) of
91
Saccharomyces.cerevisiae (Invitrogen, no. 95702, ATCC 201388), constructed by
92
oligonucleotide-directed homologous recombination to tag each open reading frame (ORF) with
93
Aequrea victoria GFP (S65T) in its chromosomal location at the 3’ end, 42 covering all the seven
94
known DNA damage repair pathways. The library expresses full-length, chromosomally tagged
95
green fluorescent protein fusion proteins, 42 which makes the GFP signal reflect protein
96
expression directly.
97
Details of the proteomics assay for using GFP-tagged yeast cells were described in our
98
previous reports. 38-41 Briefly, the yeast strains were grown in clear bottom black 384-well plates
99
(Costar) with Synthetic Dextrose base (SD base) with Dropout (DO) supplement –His (Clontech,
100
CA, US) for 4-6 h at 30 °C to reach early exponential growth. 10µL DBP sample aliquots in PBS
101
or control (PBS only) were added to each well to obtain the target concentrations (Table S1). The
102
plates were then placed in a Micro plate Reader (Synergy TM H1 Multi-Mode, Biotech, Winooski,
ACS Paragon Plus Environment
Page 6 of 34
Page 7 of 34
Environmental Science & Technology
103
VT) for absorbance (OD600 for cell growth) and GFP signal (filters with 485-nm excitation and
104
535-nm emission for protein expression) measurements every 5 min for 2 hours after double
105
orbital shake (425 cpm) for 1 minute. All tests were performed in dark in triplicate. Considering
106
that all the DBPs were tested at concentrations much lower than their solubility, the evaporation
107
and loss of the volatile DBPs during the 2-hr assay was not considered in this study. 43,44
108 109
Protein Expression Profiling Data Processing and Quantitative Molecular Endpoints
110
Derivation
111
Temporal protein expression profiling data of yeast library were processed as described
112
previously. 39, 41, 45 Temporal OD and GFP raw data are first corrected by background OD and
113
GFP signal of blank media control with or without chemical, respectively. The protein
114
expression level P for each protein biomarker (ORF) i, in treatment x, and at time point t is
115
normalized by cell density as: Pi,x,t = (GFPi,x,t-corrected/ODi,x,t-corrected) (1)
116
Where, GFPi,x,t-corrected is the GFP reading of protein i, in treatment x, at time t, corrected
117 118
by the GFP reading in the blank media control at time t; ODi,x,t-corrected is the OD reading of
119
protein i, in treatment x, at time t, corrected by the OD reading in the blank media control at time
120
t.
121
The altered protein expression in relative to untreated control (without chemical) for a
122
given protein ORFi in treatment x, at time t, due to chemical exposure, also referred as induction
123
factor I, is calculated as:
124
Ii,x,t = Pi,x,t /Pi,untreated,t (2)
ACS Paragon Plus Environment
Environmental Science & Technology
125
Where, Pi,x,t = (GFPcorrected/ODcorrected)treatment x , is the altered protein expression GFP level
126
for protein (ORF) i for treatment x, at time t, in the treated experimental condition with chemical
127
exposure; Pi,untreated,t = (GFPcorrected/ ODcorrected) untreated control, is the altered protein expression GFP
128
level for protein (ORF) i for treatment x, at time t, in the un-treated control without chemical
129
exposure. P values of both treated experiments and untreated controls are normalized against
130
internal control (housekeeping protein PGK146).
131
To quantify the chemical-induced protein expression level changes of a treatment, Protein
132
Effect Level Index (PELI) was derived as a quantitative molecular endpoint. 38-41 The
133
accumulative altered protein expression change over the 2-hr exposure period for a given protein
134
(ORF) i was calculated as:
, =
135 136 137 138 139
142 143
!"# %
(3)
Where, t is the exposure time. For up-regulated protein, I (up-regulated) = I, when I≥1; for proteins that showed down-regulation, I (up-regulated) =1 when I
