Induction of Mammalian Cell Chronic
Cytotoxicity and Acute Genomic DNA
Damage by Drinking Water
Disinfection Byproducts
Michael J. Plewa, Elizabeth D. Wagner
University of Illinois at Urbana-Champaign
Illinois Water Conference 2008
Safe Drinking Water: Benefits and Risks
6 Drinking water disinfection was a major
public health triumph of the 20th century.
The disinfectants greatly reduced the
incidence of typhoid, cholera and other
waterborne diseases.
6 Each day water utilities in the U.S.A.
produce over 1.3×1010 liters of high quality,
safe drinking water to over 270 million
people.
Richardson SD, Simmons JE, Rice G. 2002. Disinfection by-products: the next generation. Environ Sci Technol 36:1198A-1220A.
2
Safe Drinking Water: Benefits and Risks
6However, there is an unintended
consequence of disinfection, the
generation of disinfection
by-products (DBPs).
6 DBPs are toxic compounds formed
during drinking water disinfection
as a result of the reaction between
organic materials and disinfectants.
3
After 34 Years How Can DBPs
Be Emerging Contaminants?
6 Humic/fulvic structure is
undefined; you cannot
predict DBPs to close the
TOX gap.
6 Analytical chemists
dream!
– Difficult search to identify
missing DBPs
– >600 DBPs identified
– Regulatory nightmare
Unknown 69.9%
HNMs 0.5%
HACEs 0.5%
HKs 0.9%
HALDs 1.8%
HANs 0.8%
HAAs 11.8%
THMs 13.5%
Halofuranones 0.1%
IodoTHMs 0.2%
Summary distribution of DBP chemical classes in water analyzed
in the U.S. EPA Nationwide Occurrence Study as a component
of TOX. Data summarized by S. Krasner.
4
After 34 Years How Can DBPs
Be Emerging Contaminants?
6 Decreasing supplies of pristine waters →
use of impaired waters
– Algal impacts
– Wastewater impacts and wastewater use (CA
and FL)
– Use of impaired waters → more organic-N →
Nitrogenous DBP precursors
Schreiber IM and Mitch WA. 2006. Occurrence and fate of nitrosamines and nitrosamine precursors in wastewater impacted surface
waters using boron as a conservative tracer. Environ Sci Technol 40:3203-3210.
5
After 34 Years How Can DBPs Be
Emerging Contaminants?
6 U.S. EPA Stage 2 DBP Rule to reduce THMs/HAAs
– Utilities switching to ozonation and chloramination.
– Unexpected consequences → Promote N-DBP and Iodo-DBP
formation.
6 U.S. EPA and AWWA recommend more research in NDBPs, I-DBPs, and I-N-DBPs.
– EPA Workshop 2005; AWWA meeting 2005, 2006; Gordon
Research Conference 2006.
– The first comprehensive review of DBP occurrence, genotoxicity
and carcinogenicity: Mutation Research, 2007.
Richardson SD, Plewa MJ, Wagner ED, Schoeny R, DeMarini DM. 2007. Occurrence, genotoxicity, and
carcinogenicity of emerging disinfection by-products in drinking water: a review and roadmap for research.
Mutation Res 636:178-242 .
[email protected]
6
Solutions to the Impediments for a
Comparative Toxicology of DBPs
6 Structure-Function Activity studies to
define high priority DBPs.
6 EPA Nationwide Occurrence Study.
6 New analytical biological tools to integrate
analytical toxicology with the analytical
chemistry of DBPs.
Woo Y-T, Lai D, McLain JL, Manibusan MK, Dellarco V. 2002. Use of mechanism-based structure-activity relationships analysis in carcinogenic
potential ranking for drinking water disinfection by-products. Environ Health Perspect 110 (Supp 1):75-87.
Weinberg HS, Krasner SW, Richardson SD, Thruston, AD, Jr. 2002. The Occurrence of Disinfection By-Products (DBPs) of Health Concern in
Drinking Water: Results of a Nationwide DBP Occurrence Study, U.S. Environmental Protection Agency, National Exposure Research
Laboratory, Athens, GA. EPA/600/R-02/068;www.epa.gov/athens/publications/ EPA_600_R02_068. pdf
Plewa MJ, Kargalioglu Y, Vankerk D, Minear RA, Wagner ED. 2002. Mammalian cell cytotoxicity and genotoxicity analysis of drinking water
disinfection by-products. Environ Molec Mutagenesis 40:134-142.
7
Research Objectives
6 Obtain samples (~50 mg) of synthesized, analytical
grade DBPs from U.S. EPA.
6 Analyze the direct-acting cytotoxicity and genomic
genotoxicity of the individual DBPs with Chinese
hamster ovary (CHO) cells.
6 Determine the cytotoxic and genotoxic rank order of
the DBPs.
6 Develop a quantitative and comparative DBP toxicity
database.
Plewa MJ, Wagner ED. 2004. Quantitative Comparative Mammalian Cell Cytotoxicity and Genotoxicity of Selected Classes of
Drinking Water Disinfection By-Products. AwwaRF Grant #3089.
8
Chinese Hamster Ovary Cells
CHO Cells, AS52, Clone 11-4-8
6 CHO cells are widely used in toxicology
research.
6 Clone 11-4-8 was isolated by our
laboratory and expresses a stable
chromosome complement.
6 CHO AS52 cells express functional p53
protein and are competent for DNA repair.
6 The cells exhibit normal morphology,
express cell contact inhibition and grow as
a monolayer without expression of
neoplastic foci.
Wagner ED, Rayburn AL, Anderson D, Plewa MJ. 1998. Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and
forward mutation in an isolated clone of Chinese hamster ovary cells. Environ Molec Mutagenesis 32:360-368.
Rundell MS, Wagner ED, Plewa MJ. 2003. The comet assay: Genotoxic damage or nuclear fragmentation? Environ Molec Mutagenesis
42:61-67.
9
Mammalian Cell Chronic
Cytotoxicity Assay
6 Standard plating methods to measure toxicity
are laborious, time consuming, expensive and
require large amounts of sample.
6 We developed a rapid, semi-automated,
microplate-based, chronic cytotoxicity assay
that measures the impact of a specific DBP on
CHO cells to grow over a 72 h (~3 cell cycles)
period.
Plewa MJ, Kargalioglu Y, Vankerk D, Minear RA, Wagner ED. 2000. Development of quantitative comparative cytotoxicity and
genotoxicity assays for environmental hazardous chemicals. Water Sci Technol 42:109-116.
10
Cytotoxicity Absorbancy Data:
Dibromoacetamide
Wash cells, fix with MeOH,
stain, wash and scan on
microplate reader at 595 nm.
Blank correct data
blank
control
Normalize data to a percentage of
the concurrent negative control
DBP concentrations
Save data as a spreadsheet file
11
CHO Cell Cytotoxicity: Mean Cell Density
as the Percent of the Negative Control (±SE)
CHO Cell Chronic Cytotoxicity of
Dibromoacetamide: %C½ Value
100
The %C½ value is the
concentration of each
test agent that reduced
the CHO cell density by
50% as compared to
the negative control.
75
R 2 = 0.99
Br
50
Br
C
O
C
NH2
H
25
The %C½ value is
analogous to the LC50
measurement.
Dibromoacetamide
%C½ Value = 12.2 µM
0
0.1
1
10
100
Dibromoacetamide (µM)
12
CHO Cell Cytotoxicity: Mean Cell Density
as the Percent of the Negative Control
Comparative Cytotoxicity for the
Haloacetamides
100
80
60
40
20
0
DIAcAm
IAcAm
BIAcAm
CIAcAm
BAcAm
DBAcAm
TBAcAm
BCAcAm
DBCAcAm
BDCAcAm
CAcAm
DCAcAm
TCAcAm
10-7
10-6
10-5
10-4
10-3
10-2
Haloacetamides (M)
Plewa, M. J.; Muellner, M. G.; Richardson, S. D.; Fasano, F.; Buettner, K. M.; Woo, Y. T.; McKague, A. B.; Wagner, E. D.,
Occurrence, synthesis and mammalian cell cytotoxicity and genotoxicity of haloacetamides: An emerging class of nitrogenous
drinking water disinfection by-products. Environ. Sci. Technol. 2008, 42, (3), 955-961.
13
10-6
10-5
Dibromoacetamide
BDCNM
3,3-Dibromo-4-oxopentanoic Acid
Bromochloroacetamide
DBNM
DBCNM
BNM
Bromodichloroacetamide
Bromochloroacetonitrile
TBNM
Dibromoacetonitrile
Bromoacetonitrile Tribromoacetamide
Iodoacetonitrile
Bromoiodoacetamide
Tribromoacetaldehyde
Chloroacetaldehyde
Dibromochloroacetamide
Dibromoacetaldehyde
Chloroiodoacetamide
10-4
Tribromopyrrole
CNM
TCNM
Bromate
Trichloroacetaldehyde
10-3
CHO Cell Cytotoxicity as %C½ Values (~LC50)
Log Molar Concentration (72 h Exposure)
DCAA
Chloroform
Bromodichloromethane
Bromoform
2-Bromo-3-methylbutenedioic Acid
Chlorodibromomethane
EMS +Control
Tribromopropenoic Acid
Dibromoiodomethane
Dichloroacetamide
2-Bromobutenedioic Acid
2,3-Dibromopropenoic Acid Trichloroacetamide
TCAA
Bromochloroiodomethane
BDCAA
BIAA
BCAA
CAA 2-Iodo-3-methylbutenedioic Acid
DBAA
3,3-Dibromopropenoic Acid
DIAA
DCNM
Chloroacetamide
Trichloroacetonitrile
3-Iodo-3-bromopropenoic Acid
DBCAA
MX
TBAA
Dichloroacetonitrile
Chloroacetonitrile
Iodoform
3,3-Bromochloro-4-oxopentanoic Acid
Dichloroacetaldehyde
BCNM
2-Iodo-3-bromopropenoic Acid
BAA
IAA
Bromoacetamide
Iodoacetamide
Diiodoacetamide
Comparative DBP CHO Cell Chronic
Cytotoxicity Database
DBP Chemical Class
Other DBPs
Haloacetamides
Haloacetaldehydes
Haloacetonitriles
Halomethanes
Halonitromethanes
Halo Acids
Haloacetic Acids
10-2
March 2008
14
Genomic DNA Damage Induced by
Drinking Water Disinfection By-Products
Single Cell Gel
Electrophoresis
The target is the genome,
not just a gene.
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu J-C, Sasaki YF. 2000. Single cell
gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Molec Mutagenesis 35:206-221.
15
Single Cell Gel
Electrophoresis
• CHO cells grown in microplate wells
were treated with a test agent for 4h.
• The cells were harvested and placed in
an agarose sandwich upon a SCGE
microscope slide – the microgel.
• The cell membranes were lysed and
the DNA in the nuclei were denatured.
• The naked nuclei were electrophoresed
at a pH >13.5.
• The microgels were neutralized and
stained with ethidium bromide.
• Each microgel was analyzed with a
fluorescence microscope with a CCD
computer interface.
• The data were stored in computer
spreadsheets.
16
Computer Analysis of SCGE Images
• The nuclei were analyzed with a Zeiss fluorescence microscope using an
excitation filter of BP 546/10 nm and a barrier filter of 590 nm. A
computerized image analysis system was employed to measure various
Comet parameters. (Komet 3.1, Kinetic Imaging Ltd).
• The tail moment is the integrated value of DNA density multiplied by the
migration distance. The % tail DNA is the amount of DNA that has migrated
into the gel from the nucleus.
.
17
Acute Cytotoxicity
6 From each treated cell
suspension a 10 µl aliquot
was stained with 10 µl of
0.05% trypan blue vital dye
in PBS.
6 The percent survival for
each treatment group was
determined by counting the
dead cells (blue) and the
live cells (clear).
18
100
90
80
70
Control
30 µM
DBNM
40 µM
DBNM
Average Median SCGE Tail Moment ±SE
60
50
80
Acute Cytotoxicity
% Viable Cells
Genomic DNA Damage
Induced by
Dibromonitromethane
Plewa MJ, Wagner ED, Jazwierska P, Richardson SD, Chen PH, McKague AB. 2004.
Halonitromethane drinking water disinfection by-products: chemical characterization and
mammalian cell cytotoxicity and genotoxicity. Environ Sci Technol 38:62-68.
Br
Br
C
NO2
60
H
40
20
SCGE Genotoxic
Potency = 26.2 µM
0
0
10
20
30
40
50
Dibromonitromethane (µM)
19
CHO Cell Genomic DNA Damge as the
Average Median SCGE Tail Moment Value
Comparative Genotoxicity of the
Haloacetamides
120
100
80
60
40
IAcAm
DIAcAm
BIAcAm
CIAcAm
BAcAm
DBAcAm
TBAcAm
BCAcAm
DBCAcAm
BDCAcAm
CAcAm
DcAcAm
TCAcAm
20
0
10-5
10-4
10-3
10-2
Haloacetamides (M)
Plewa, M.J.; Muellner, M.G.; Richardson, S.D.; Fasano, F.; Buettner, K.M.; Woo, Y.T.; McKague, A.B.; Wagner, E.D. Environ. Sci. 20
Technol. 2008, 42, (3), 955-961.
10-6
10-5
Haloacetamides
Haloacetaldehydes
Haloacetonitriles
DBNM
Iodoacetamide
Dibromoacetonitrile Diiodoacetamide
Iodoacetonitrile
Tribromoacetamide
Bromoacetonitrile Bromoacetamide
Other DBPs
Halonitromethanes
10-4
BNM
DBCNM
BCNM
Bromodichloroacetamide
Chloroacetaldehyde
Dibromoacetaldehyde
10-3
Not Genotoxic: DCAA, TCAA, BDCAA, Dichloroacetamide, Chloroform, Chlorodibromomethane, Bromoform, Iodoform,
Bromochloroiodomethane, Dibromoiodomethane, Bromodichloromethane, 3,3-Dibromopropenoic Acid,
3-Iodo-3-bromopropenoic Acid, 2,3,3,Tribromopropenoic Acid, trans-2-bromo-3-methylbutenedioic Acid, Trichloroacetaldehyde
CNM
Dichloroacetonitrile
Chloroacetamide
CDBAA
2-Bromobutenedioic Acid
EMS +Control
2-Iodo-3-methylbutenedioic Acid
Trichloroacetamide
2-Iodo-3-bromopropenoic Acid
Bromate
2,3-Dibromopropenoic Acid
TBAA
BIAA
BCAA
DBAA
DIAA
Chloroacetonitrile Bromochloroacetamide
Dibromoacetamide
Dichloroacetaldehyde
Trichloroacetonitrile
MX
Chloroiodoacetamide
Tribromopyrrole
Bromochloroacetonitrile
3,3-Bromochloro-4-oxopentanoic Acid
Tribromoacetaldehyde
CAA
DCNM
BAA
DBP Chemical Class
BDCNM
Dibromochloroacetamide
TBNM
Bromoiodoacetamide
3,3-Dibromo-4-oxopentanoic Acid
TCNM
Halo Acids
IAA
Comparative DBP CHO Cell Acute
Genotoxicity Database
Haloacetic Acids
10-2
Single Cell Gel Electrophoresis Genotoxicity Potency Values
Log Molar Concentration (4 h Exposure)
March 2008
21
Comparison Between CHO Cell
Cytotoxicity and Genotoxicity of DBPs
SCGE Genotoxic Potency Value (M)
10-1
10-2
10-3
10-4
10-5
r = 0.58; P< 0.001
10-6
10-7
10-6
10-5
10-4
10-3
6 A large dataset was available
for a correlation test.
6 This consisted of six chemical
classes of DBPs and related
compounds that induced
significant chronic cytotoxicity
and acute genotoxicity.
6 A direct, highly significant (P ≤
0.001) correlation was observed
(r = 0.58).
10-2
%C1/2 Cytotoxic Potency Value (M)
Plewa, M. J.; Wagner, E. D., Quantitative Comparative Mammalian Cell Cytotoxicity and Genotoxicity of Selected
Classes of Drinking Water Disinfection By-Products. Am. Water Works Res. Foundation: Denver, CO, 2008 (In Press).
22
Conclusions I
6 With our current database, ~70 DBPs were
compared on a level toxicological playing field.
6 We can quantitatively compare the cytotoxicity of
DBPs using their %C½ values.
6 We can quantitatively compare the genotoxicity of
DBPs using the SCGE Genotoxic Potency values.
6 We can compare classes or specific groups of
DBPs based on their Toxicity Index. Within a
class, the reciprocal of the averaged median %C½
values is the cytotoxicity index value and the
reciprocal of the averaged median genotoxic
potency values is the genotoxicity index value.
23
Comparison of the Cytotoxicity and
Genotoxicity of DBP Classes
Cytotoxicity
Genotoxicity
DBP Chemical Class
Halomethanes
Haloacetic Acids
5 Haloacetic Acids
>2C Haloacids
Haloacetonitriles
Haloacetamides
Halonitromethanes
Haloacetaldehydes
102
103
104
105
CHO Cell Cytotoxicity or Genotoxicity Index Values (log scale)
24
Toxicity Index of DBP Classes:
(impact of the halogen leaving group)
DBP Chemical Class
Cytotoxicity
Genotoxicity
Chloro-DBPs
Bromo-DBPs
Iodo-DBPs
March 2007
102
103
104
105
CHO Cell Cytotoxicity or Genotoxicity Index Values (log scale)
25
CHO Cell Cytotoxicity or
Genotoxicity Index Values (log scale)
Toxicity Index of DBP Classes:
(C-DBPs versus N-DBPs)
105
Cytotoxicity
Genotoxicity
104
103
102
Carbon-Based DBPs
Nitrogen-Containing DBPs
26
Conclusions II
6 The current U.S. EPA-regulated DBP classes
(THMs and HAAs) are substantially less toxic
than emerging DBPs.
6 Iodinated-DBPs are far more toxic than their
brominated and chlorinated analogs.
6 N-DBPs are much more toxic than C-DBPs.
6 The occurrence of these emerging DBPs are on
the rise because of changes in source water
quality and the increased use of alternative
water disinfectants.
6 These emerging DBPs may pose adverse health
risks.
27
Future
Future Studies
Studies
6 Expand the number of DBP classes
analyzed (cyanogen halides, aldehydes
ketones, nitrosamines etc.)
6 Use the database to guide the analysis of
drinking water for emerging DBPs.
6 Improve the resolution of the in vitro
toxicity assays and determine the
mechanism of toxic action by the use of
human toxicogenomic analysis.
28
Students and Colleagues
6
6
6
6
6
6
6
Dr. Yahya Kargalioglu
Dr. Mark Muellner
Ms. Nancy Hsu
Dr. Eduardo Cemeli
Mr. Justin Pals
Ms. Jessica Wallace
Ms. Yukako Kumada
6
6
6
6
6
Dr. Susan Richardson
Dr. Yin-Tak Woo
Dr. Bruce McKague
Dr. Matt Hudson
Dr. Benito Mariñas
Funded in part by AwwaRF Grants 554 & 3089, U.S. EPA Grant CR83069501, IllinoisIndiana Sea Grant R/WF-09-06, and WaterCAMPWS NSF Center Grant CTS-0120978.
M. Muellner was supported by T32 ES07326 NIH Predoctoral Fellowship.
29