mammalian cell cytotoxicity and - us epa · mammalian cell cytotoxicity and genotoxicity of new...

EPA Region 5July 2004

1

Mammalian Cell Cytotoxicity and Genotoxicity of New Drinking Water

Disinfection By-Products

U.S. EPA Region 5 ORD STAR Seminar

Dr. Michael J. PlewaProfessor of Genetics

University of Illinois at Urbana-Champaign


2

Water is the best of all things. Pindar (438 BC), Olympian Odes


3


4

Safe Drinking Water:Benefits and Risks

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. However, there is an unintended consequence of disinfection, the generation of chemical disinfectionby-products (DBPs).


5

Drinking Water Disinfection By-Products (DBPs)

• DBPs are compounds formed during drinking water disinfection as a result of the reaction between naturally occurring organic materials, synthetic organic contaminants and disinfectants.

• Between 51% and 92% of DBP products are unknown in the halogenated organic fraction (TOX) depending on the disinfection process.


6

Regulation of DBPs

• The health risks due to DBPs are not fully known, however, a substantial number of these agents were demonstrated to be toxic in many biological assays. In 1979 the EPA began the formal regulation of DBPs.

• The regulation was extended in 1998 with the publication of the Stage 1 Disinfectants/Disinfection Byproducts Rule.

• Stage 2 of the Rule is in the process of finalization. • Although over 600 DBPs have been isolated and

identified, this represents only a fraction of the halogenated organic material that can be isolated after the disinfection of raw waters.


7

No DBP Toxicity Database• In 1999 the U.S. EPA, the National Institute of Environmental

Health Sciences and the U.S. Army called for a comprehensive biological and mechanistic DBP database.

• EPA employed a computer model-based structure-activity relationship (SAR) analysis of hundreds of DBPs. The SAR analysis was used to rank the carcinogenic potential of DBPsand identify a group of priority DBPs for further chemical and biological analysis.

• The EPA also conducted a Nationwide DBP Occurrence Study. A list of priority DBPs was drawn up of approximately 50 agents that were not included in the Information Collection Rule and were estimated to be the most toxic. A second group of approximately 20 DBPs of similar chemical structure to the priority compounds was also defined. Also from the surveyed water treatment plants, 28 new DBPs were structurally identified.

• There is virtually no toxicity data for most of these priority and new DBPs.


8

DBP Ignorance

Unknown 69.9%

THMs 13.5%

HAAs 11.8%

Halofuranones 0.1%IodoTHMs 0.2%

HANs 0.8%HALDs 1.8%HKs 0.9%HACEs 0.5%HNMs 0.5%

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 Dr. S. Krasner.


9

In a Recent Issue of theJournal Epidemiology

• A panel of international experts stated, “These findings strengthen the hypothesis that the risk of bladder cancer is increased with long-term exposure to disinfection byproducts at levels currently observed in many industrialized countries.”

• Epidemiology 2004, 15:357-367


10

Objectives of This Study

• Analyze the cytotoxicity of the individual DBPs with Chinese hamster ovary (CHO) cells.

• Determine the cytotoxic rank order of the DBPs.• Analyze the genotoxicity of the individual DBPs

with CHO cells.• Determine the genotoxic rank order of the DBPs.• Employ these data in a U.S. Environmental

Protection Agency risk assessment program.


11

Mammalian Cell Chronic Cytotoxicity Assay

Chronic mammalian cell cytotoxicity is an important measure of the toxic impact of a test agent in which cells are continuously exposed throughout several cell divisions. Standard plating methods to measure toxicity are laborious, time consuming and require large amounts of sample. To address these problems we developed a rapid, semi-automated microplate-based, chronic cytotoxicity assay that measured the impact of a specific water DBP on cell survivorship.


12

Mammalian Cell ChronicCytotoxicity Assay

CHO Cells per Microplate Well0 20000 40000 60000 80000C

HO

Cel

l Den

sity

as

Abs

orba

ncy

at 5

95 n

m0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Wavelength (nm)400 600 800

CV

Abs

orba

ncy

0.00

0.05

0.10

0.15

0.20 A

B

• CHO cells were exposed to a known concentration of a DBP in a microplate well for 72 h in a CO2 incubator at 37°C.

• After incubation the cells were fixed, stained with crystal violet, washed and 50 µl DMSO was added to each well and analyzed with a microplate reader at 595 nm.

• The data were transferred onto an Excel spreadsheet and analyzed.

• The absorbancy and the cell density were significantly and highly correlated (r = 0.98, P < 0.001). (A) Absorption spectrum of crystal violet in the range

from 340 to 800 nm. (B) A comparison of the number of cells per microplate well determined with a Coulter counter and the absorbancy of identical wells after crystal violet staining.


13

Determination of Optimal CHO Cell Plating Density

Original Cell Titer Plated

1000 2000 3000 4000 5000 6000

Mea

n Pe

rcen

t of C

HO

Cel

l Gro

wth

of t

heth

e 30

00 C

ell T

iter C

ontro

l Afte

r 72

h (±

SE)

20

40

60

80

100

120

140

3000 CHO cells plated will grow to near confluency after 72h.


14

CHO Cell Cytotoxicity of Dibromoacetic Acid: %C½ Value

Dibromoacetic Acid (µM)

0 200 400 600 800 1000

CH

O C

ell C

ytot

oxic

ity: C

ell D

ensi

tyas

a %

of t

he N

egat

ive

Con

trol (

±SE

)

0

25

50

75

100

%C1/2 = 521.4 µM

r2 = 0.99

The %C½ value is the concentration of each test agent that reduced the CHO cell density by 50% as compared to the negative control.

The %C½ value is analogous to the LC50measurement.


15

Haloacetic Acids: Comparison of CHO Cell Cytotoxicity(EPA Additivity Project)

Haloacetic Acid Concentration (µM)100 101 102 103 104

CH

O C

ell C

ytot

oxic

ityM

ean

% o

f the

Neg

ativ

e C

ontro

l

0

20

40

60

80

100

120

BADBATBACADCATCA

DBCABDCA

BCA


16

CHO Cell Cytotoxicity of Halonitromethanes

Test Agent Concentration (mM)0.001 0.01 0.1 1 10

CH

O C

ell C

ytot

oxic

ityP

erce

nt o

f the

Neg

ativ

e C

ontro

lM

ean

Cel

l Den

sity

A59

5

0

20

40

60

80

100

120

Potassium bromateEthylmethanesulfonate

BromonitromethaneDibromonitromethaneTribromonitromethane

BromochloronitromethaneDibromochloronitromethaneBromodichloronitromethane

ChloronitromethaneDichloronitromethane Trichloronitromethane


17

CHO Cell Chronic Cytotoxic Potencyas %C1/2 Values (Molar Concentration)

10-6 10-5 10-4 10-3 10-2

Drin

king

Wat

er D

isin

fect

ion

Byp

rodu

cts

EMSBromate

CADCATCABCA

BDCADBCA

BADBATBA

IA33DBPA23DBPA

TBPABMBA

BBADBBADBOP

IBPAMX

TBPCNM

DCNMTCNMBCNM

DCBNMDBCNM

BNMDBNMTBNMComparison of DBP

Chronic Cytotoxicity to CHO Cells

The halonitromethaneswere more cytotoxic to mammalian cells than the haloacetic acids.

The brominated HNMsand HAAs were more cytotoxic than their chlorinated analogs.


18

DBP-Induced Mammalian Cell Cytotoxicity: Summary

• Our microplate-based method allows for the analysis of a large number of concentrations per test agent and a large number of replicates per concentration.

• Chronic mammalian cell cytotoxicity is an important toxicological measurement and is being used for risk assessment by the US EPA.

• The cytotoxic potency (%C½ value) permits a quantitative comparison and rank ordering of the DBPs.

• The HNMs were more cytotoxic than HAAs.• Brominated HNMs and HAAs were more cytotoxic than

their chlorinated analogs.


19

Genomic DNA Damage Induced by Drinking Water

Disinfection By-Products

Single Cell Gel Electrophoresis

The target is the genome, not just a gene.


20

DNA Damage


21

SCGE Analysis of 2AAAF(in isolated nuclei from control and treated CHO cells)

Negative ControlNegative Control 800 800 nMnM 2AAAF2AAAF


22

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. • The tail moment is the integrated value of DNA density multiplied by the migration distance.

..


23

Acute Cytotoxicity• From each treated cell

suspension a 10 µl aliquot was stained with 10 µl of 0.05% trypan blue vital dye in PBS.

• The percent survival for each treatment group was determined by counting the dead cells (blue) and the live cells (clear).


24

Genomic DNA Damage Inducedby Dibromonitro-

methane

Dibromonitromethane (µM)0 10 20 30 40 50

Ave

rage

Med

ian

SC

GE

Tai

l Mom

ent ±

SE

0

20

40

60

80

Acute C

ytotoxicity%

Viable C

ells

50

60

70

8090100

Control

30 µMDBNM

40 µMDBNM


25

SCGE Analysis ofHaloacetic Acids in CHO Cells

Haloacetic Acid mM0.001 0.01 0.1 1 10

Aver

age

Med

ian

SCG

E T

ail M

omen

t

0

10

20

30

40

50

60

70

80

90

B

BADBATBACADCATCAMX

EMSKBrO3


26

SCGE Analysis ofHalonitromethanes in CHO Cells

Concentration (mM)0.01 0.1 1 10

Ave

rage

Med

ian

SC

GE

Tai

l Mom

ent

0

10

20

30

40

50

60

70

80

90

DibromonitromethaneBromonitromethane

Tribromonitromethane

BromochloronitromethaneDibromochloronitromethaneBromodichloronitromethane Trichloronitromethane

DichloronitromethaneChloronitromethane Ethylmethanesulfonate

KBrO3


27

DBP CHO Cell Genotoxicity

CHO Cell SCGE Acute Genotoxic Potency (Molar Concentration)

10-5 10-4 10-3 10-2

Drin

king

Wat

er D

isin

fect

ion

Byp

rodu

cts

EMSBromate

CADCATCABCA

BDCADBCA

BADBATBA

IA33DBPA23DBPA

TBPABMBA

BBADBBADBOP

IBPAMX

TBPCNM

DCNMTCNMBCNM

DCBNMDBCNM

BNMDBNMTBNM

NSNS

Experiments in progressExperiments in progressExperiments in progressExperiments in progressExperiments in progress

Experiments in progress

Experiments in progressExperiments in progressExperiments in progress

Experiments in progress

In general, the halonitromethaneswere more genotoxic to CHO cells than the haloacetic acids.The brominated HNMsand HAAs were more genotoxic than their chlorinated analogs.


28

Newly Identified DisinfectionBy-Products (2002-2004)

N

BrBr

HBr

H

2,3,5-Tribromopyrrole

C C

O

OH

H

I

H H

Cl

H

OH

O

C C

H

Br

H

OH

O

C C

Iodoacetic acid Bromoacetic acid Chloroacetic acid


29

Trbrpy_b #217-238 RT: 8.52-8.87 AV: 22 SB: 101 7.55-8.18, 9.05-10.10 NL: 8.71E5T: + c Full ms [ 45.00-450.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

302.9

224.0

222.1226.0 301.0

306.8197.0

195.1

144.1118.163.1227.0 307.8115.1 161.279.1 300.0 446.1401.0342.0 358.8

Mass Spectra of 2,3,5-Tribromopyrrole and

Iodoacetic Acid

Tribromopyrrole

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220m/z0

100

%

73

42

345958

5150 65

61 68

200

141

127

10878 8582 9389 109140

128

169

168142 201

CH

O3

52

(-I)

HCH

I 2-C(O)OCH3

CH

OI

22

-OCH3

CH

OI

35

2

Iodoacetic acid methyl ester


30

CHO Cell Chronic Cytotoxicity of Tribromopyrrole

Tribromopyrrole (µM) 72 h Treatment

0 20 40 60 80 100

CH

O C

ell C

ytot

oxic

ity: C

ell D

ensi

tyas

a %

of t

he N

egat

ive

Con

trol (

±SE

)

0

25

50

75

100

%C½ = 60.6 µM (r2 = 0.96)

• In the chronic CHO cell cytotoxicity assay the level of cell killing by tribromopyrroleduring the 72 h period was similar to that of BCNM and TBA.

• The %C½ value was 60.6 µM.


31

CHO Cell Chronic Cytotoxicity of Iodoacetic Acid

Iodoacetic Acid (µM) 72 h Treatment

0.1 1 10

CH

O C

ell C

ytot

oxic

ity: C

ell D

ensi

tyas

a P

erce

nt o

f the

Neg

ativ

e C

ontro

l

0

20

40

60

80

100• Iodoacetic acid was the most potent cytotoxic DBP among the 30 analyzed in our laboratory.

• The %C½ value for iodoacetic acid was 2.9 µM.


32

Rank Order of DBP CHO Cell

Cytotoxicity

DBP %C1/2 mMDBNM 0.006BNM 0.007DBCNM 0.007TBNM 0.008BA 0.010BDCNM 0.013BCNM 0.041TBA 0.085MX 0.275DCNM 0.373DBA 0.521CNM 0.529TCNM 0.536CA 0.848PB 0.963TCA 2.400EMS 4.250DCA 7.304

IA = 0.003

The halonitromethaneswere more cytotoxic to mammalian cells than the haloacetic acids.

The brominated HNMsand HAAs were more cytotoxic than their chlorinated analogs.

TBP =0.06


33

Genotoxicity of Tribromopyrrole

Tribromopyrrole (µM) 4 h Treatment0 100 200 300

SC

GE

Med

ian

Tail

Mom

ent (

±SE)

0

10

20

30

40

50 Mean S

CG

E %

Tail DN

A (±S

E)

0

10

20

30

40

50

60

70SCGE Tail MomentSCGE % Tail DNA• Tribromopyrrole

is a strong genotoxic agent in CHO cells.

• The TM SCGE genotoxic potency of TBP is 301.5 µM.


34

Genotoxicity of Iodoacetic Acid

Iodoacetic Acid (µM)

0 5 10 15 20

Aver

age

Med

ian

Tail

Mom

ent (

±SE)

0

10

20

30

40

50

60

Average SCG

E % Tail D

NA (±SE)

0

10

20

30

40

50

60

70

• Iodoacetic acid is the most potent genotoxic DBP (of 22) that we analyzed in CHO cells.

• The TM SCGE genotoxic potency of iodoacetic acid is 7.9 µM.


35

Rank Order of DBP CHO Cell Genotoxicity

DBP CHOGenotoxic

Potency (mM)

BA 0.017

DBNM 0.026

TBNM 0.070

TCNM 0.093

BNM 0.133

DBCNM 0.143

BCNM 0.166

MX 0.244

CA 0.411

BDCNM 0.413

DCNM 0.420

DBA 1.756

TBA 2.456

CNM 3.007

EMS 6.057

KBrO37.195

DCA NS

TCA NS

IA = 0.008

In general, the halonitromethaneswere more genotoxic to CHO cells than the haloacetic acids.The brominated HNMsand HAAs were more genotoxic than their chlorinated analogs.

TBP = 0.301


36

Newly Identified DisinfectionBy-Products (2002-2004)

N

BrBr

HBr

H

2,3,5-Tribromopyrrole

C C

O

OH

H

I

H H

Cl

H

OH

O

C C

H

Br

H

OH

O

C C

Iodoacetic acid Bromoacetic acid Chloroacetic acid


37

Comparative Cytotoxicity of the Monohalogenated Acetic Acids

• All of the mono-halogenated acetic acids were chronic toxins to CHO cells.

• The %C½ values for iodo-, bromo-, and chloroacetic acid were 2.9, 10.0, and 848 µM, respectively.

Haloacetic Acid (µM)

0.1 1 10 100 1000

CH

O C

ell C

ytot

oxic

ity: C

ell D

ensi

tyas

a %

of t

he N

egat

ive

Con

trol

0

20

40

60

80

100

Iodoacetic AcidBromoacetic AcidChloroacetic Acid


38

Comparative Genotoxicity of the Monohalogenated Acetic Acids

Haloacetic Acid µM

1 10 100 1000

Aver

age

Med

ian

SCG

E Ta

il M

omen

t

0

10

20

30

40

50

60

70Iodoacetic AcidBromoacetic AcidChloroacetic Acid

• All of the mono-halogenated acetic acids were acute genotoxins to CHO cells.

• The SCGE genotoxic potencies for iodo-, bromo- and chloro-acetic acid were 7.9, 17, and 411 µM, respectively.


39

DBP-Induced Mammalian Cell Genotoxicity: Summary

• The mammalian cell microplate SCGE method allows for the quantitative genotoxic analysis of small amounts of test agent.

• In general the HNMs are more genotoxic than HAAs.• The genotoxic potency for both the HNMs and HAAs

is highest for the brominated analogs, followed by the bromo-chloro analogs, and then the chlorinated analogs.

• New DBPs, such as tribromopyrrole and iodoaceticacid, can be rapidly analyzed for their cytotoxic and genotoxic activity in mammalian cells.


40

Mechanisms of Haloacetic Acid Genotoxicity

• No DNA adducts have been identified.• Haloacetic acids (HAAs) induce mutation

in Salmonella and mammalian cells.• HAAs are good inducers of DNA strand

breaks in mammalian cells.• In rodent cancer studies HAAs caused

peroxisome proliferation and may induce oxidative stress.


41

Effect of Catalase or BHA on Modulating Iodoacetic Acid Genotoxicity

• Catalase is an enzyme that specifically degrades H2O2.

• Butylated hydroxyanisole is a potent radical scavenger.

• We treated CHO cells with iodoaceticacid and catalase or BHA and determined SCGE DNA damage.


42

Aver

age

Med

ian

SCG

E Ta

il M

omen

t (±S

E)

0

5

10

15

20

Treatment Group

Neg

ativ

e C

ontro

l

500

U/m

l Cat

alas

e

10 µ

M B

HA

50 µ

M B

HA

100

µM B

HA

25 µ

M IA

25 µ

M IA

+

500

U/m

l Cat

alas

e

25 µ

M IA

+ 1

0 µM

BH

A

25 µ

M IA

+ 5

0 µM

BH

A

25 µ

M IA

+ 1

00 µ

M B

HA

Inhibition of Iodoacetic Acid DNA Damage by Catalase or BHAExperiments 072503EC, 073003EC,080503EC, 080803EC, 081003EC, 081203EC


43

Take-Home Message

• Chemical disinfection of water reduced the incidence of waterborne diseases.

• Disinfection unfortunately generates a large number of halogenated DBPs.

• Epidemiological studies have linked consumption of water containing DBPswith enhanced risks of spontaneous abortion, birth defects and cancer.


44

Take-Home Message• The vast majority of DBPs that comprise

TOX have not been chemically or biologically characterized.

• The U.S. EPA has a high priority to identify DBPs and determine their toxicity.

• We demonstrated that CHO cell microplate cytotoxicity and SCGE assays were sensitive and accurate when working with small amounts of DBPs.


45

Take-Home Message• The halonitromethanes (HNM) were both more

cytotoxic and genotoxic as compared to the haloacetic acids.

• The brominated and iodinated HAAs and HNMswere more cytotoxic and genotoxic than their chlorinated analogs.

• HAAs induce their genotoxic damage via an oxidative stress mechanism.

• The goal is to engineer drinking water disinfection technologies that generate the lowest levels of highly toxic DBPs. Our work is a step in this direction.


46

This research was funded in part by AWWARF grant 554,U.S. EPA grants R825966, R83069501, and NSF grant INT97-26617

mammalian cell cytotoxicity and - us epa · mammalian cell cytotoxicity and genotoxicity of new...

Documents