Co- -exposure of Arsenite and exposure of Arsenite and Co - - PowerPoint PPT Presentation

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Co- -exposure of Arsenite and exposure of Arsenite and Co - - PowerPoint PPT Presentation

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Co- -exposure of Arsenite and exposure of Arsenite and Co Benzo(a)pyrene: Effect of : Effect of Benzo(a)pyrene glutathione on DNA adduct glutathione on DNA adduct levels levels 1 Glenn Talaska 1 Glenn Talaska Jay Vietas 1,2 1,2 Jay

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SLIDE 1

Co Co-

  • exposure of Arsenite and

exposure of Arsenite and Benzo(a)pyrene Benzo(a)pyrene: Effect of : Effect of glutathione on DNA adduct glutathione on DNA adduct levels levels

Glenn Talaska Glenn Talaska1

1

Jay Vietas Jay Vietas1,2

1,2 1 1Department of Environmental Health,

Department of Environmental Health, University of Cincinnati, University of Cincinnati, 2

2United States Air

United States Air Force Force

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SLIDE 2

Arsenic and Cancer Arsenic and Cancer

► ►Medicinal Uses and Occupational Exposures

Medicinal Uses and Occupational Exposures

► ►Higher dose chronic studies (> 100

Higher dose chronic studies (> 100 ug ug/L) /L)

  • Tseng

Tseng et al et al. (1968) in Taiwan . (1968) in Taiwan

  • Confirmation in other studies

Confirmation in other studies

► ►Chile, China, Bangladesh

Chile, China, Bangladesh

► ►Lower dose studies (< 100

Lower dose studies (< 100 ug ug/L) have not /L) have not

  • bserved increased cancer risk
  • bserved increased cancer risk
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SLIDE 3

Occupational Epidemiology Occupational Epidemiology

► ►Occupational Exposures

Occupational Exposures

  • Smelters, pesticides, fossil fuel combustion

Smelters, pesticides, fossil fuel combustion ► ►Strong relation between cancer risk and:

Strong relation between cancer risk and:

  • Length of exposure

Length of exposure

  • Amount of exposure

Amount of exposure ► ►Interaction effect of smoking and arsenic

Interaction effect of smoking and arsenic

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SLIDE 4

Modeling Arsenic Modeling Arsenic

► ►Problems with animal model

Problems with animal model

  • As not a complete carcinogen

As not a complete carcinogen

  • Cancer observed at

Cancer observed at cytotoxic cytotoxic levels levels ► ►Co

Co-

  • Carcinogenesis

Carcinogenesis

  • UV

UV

  • BaP

BaP

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SLIDE 5

Benzo(a)pyrene Benzo(a)pyrene

► ► Common component

Common component

  • f PAH mixtures: coal
  • f PAH mixtures: coal

tar, tobacco smoke tar, tobacco smoke

► ► Used to model entire

Used to model entire family of PAHs family of PAHs

► ► Exposure results in

Exposure results in formation of stable formation of stable DNA adducts DNA adducts

  • Measurable with

Measurable with sensitive assay sensitive assay

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SLIDE 6

µM As

5 10

Fold change in adduct level

1 10 100 0.1 M BaP 0.5 M BaP 1.0 M BaP

Effect of Arsenite on BaP Adducts in Effect of Arsenite on BaP Adducts in Hepa Hepa-

  • 1 Cells

1 Cells

Maier et al. (2002) Mutat. Res., 517, 101

17x

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SLIDE 7

Effect of Arsenite on Lung BaP Adducts in C57BL/6 Mice

50

As + BaP BaP Adducts per 10

9 Nucleotides

Evans et al. (2004) Carcinogenesis, 25, 493

19.9 * 12.7 * p = 0.038

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SLIDE 8

Effect of Arsenite on Skin BaP Adducts in C57BL/6 Mice

50 100 150 200 250 300 350 400 450 500

As + BaP BaP Adducts per 109 Nucleotides

Evans et al. (2004) Carcinogenesis

364.1 307.1

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SLIDE 9

Common Link Common Link

► ►Arsenite Metabolism

Arsenite Metabolism

  • Series of steps involving reduction and

Series of steps involving reduction and methylation methylation

  • Requires glutathione as a cofactor

Requires glutathione as a cofactor ► ►Benzo(a)pyrene

Benzo(a)pyrene Detoxification Detoxification

  • Glutathione conjugation

Glutathione conjugation ► ►Common biological link: Glutathione

Common biological link: Glutathione

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SLIDE 10

Potential Mechanism for BAP and As Potential Mechanism for BAP and As Interactions Interactions

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SLIDE 11

Glutathione (GSH) Glutathione (GSH)

► ► Function

Function

  • Major

Major thiol thiol in in redox redox homeostasis homeostasis

  • 1

1-

  • 10

10 mM mM in all cells in all cells

  • Conjugates with

Conjugates with electrophiles electrophiles

► ► Synthesis

Synthesis

  • Six Enzymes

Six Enzymes

  • Glutamate

Glutamate-

  • cysteine

cysteine ligase (GCL) ligase (GCL)

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SLIDE 12

Glutathione Modulation Glutathione Modulation

► ►GCL Inhibitors

GCL Inhibitors

  • Buthionine Sulfoximine (BSO)

Buthionine Sulfoximine (BSO)

  • GCLM (

GCLM (-

  • /

/ -

  • )

) ► ►Increasing GSH

Increasing GSH

  • Glutathione ethyl ester (GSH

Glutathione ethyl ester (GSH-

  • EE)

EE)

  • N

N-

  • acetyl cysteine

acetyl cysteine

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SLIDE 13

Hypothesis Hypothesis

Changes in intracellular glutathione Changes in intracellular glutathione levels are levels are inversely inversely related to related to arsenite arsenite’ ’s s effect on effect on benzo(a)pyrene benzo(a)pyrene DNA adduct levels DNA adduct levels in vitro and in in vitro and in vivo vivo. .

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SLIDE 14

Phase 1: Effect of decreased GSH Phase 1: Effect of decreased GSH In vitro In vitro

► ►Characterize dose

Characterize dose-

  • response relationship

response relationship between arsenite, BSO, and BaP DNA between arsenite, BSO, and BaP DNA adducts using HEPA adducts using HEPA-

  • 1 cells

1 cells

► ►Observe the effect of endogenous reduction

Observe the effect of endogenous reduction

  • f glutathione levels using
  • f glutathione levels using Gclm

Gclm( (-

  • /

/ -

  • )

) MEFs MEFs

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SLIDE 15

Cell Treatment Protocol Cell Treatment Protocol

0 hrs 12 24 36 48 Treat with Arsenite and BSO Add BSO Treat with BaP Harvest Cells

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SLIDE 16

BaP DNA Adduct Levels in Hepa-1 Cells Treated with Arsenite and BSO

50 100 150 200 250 300 350 400 450 500 1 5 10 15

Buthionine Sulfoximine (µM)

% BaP DNA Adducts compared to Baseline

(no arsenite or BSO treatment)

0 µM As 0.5 µM As 1 µM As 2 µM As

Baseline

Error bars represent standard error of mean

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SLIDE 17

Effect of Arsenite on BaP DNA Adducts in Gclm(+/+) and Gclm(-/-) Mouse Embryonic Fibroblasts

50 100 150 200 250 300 350 400

Gclm (+/+) Gclm (-/-)

BaP DNA Adduct Level per 10

9Nucleotides

0 uM As 0.125 uM As

Error bars represent standard error of mean

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SLIDE 18

Conclusions Conclusions – – Effect of decreased GSH Effect of decreased GSH in vitro in vitro

► ►Arsenite potentiates BaP DNA adduct levels

Arsenite potentiates BaP DNA adduct levels

► ►As GSH decreases, BaP DNA adduct levels

As GSH decreases, BaP DNA adduct levels increase increase

► ►Decreased GSH levels appear to increase

Decreased GSH levels appear to increase the cell the cell’ ’s sensitivity to arsenite s sensitivity to arsenite

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SLIDE 19

Phase 1: Effect of decreased GSH: Phase 1: Effect of decreased GSH: In vivo In vivo

► ►C57BL/6 Mice Treatments

C57BL/6 Mice Treatments

  • Arsenite in DW

Arsenite in DW

  • BSO

BSO

  • BaP

BaP ► ►Wild Type and

Wild Type and Glcm Glcm ( (-

  • /

/ -

  • )

)

  • Arsenite in DW

Arsenite in DW

  • BaP

BaP

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SLIDE 20

2 mg/L Sodium Arsenite MilliQ Water

21 days

  • f treatment

8 daily doses administered 1 hour after topical treatment of BaP

C57BL/6

BSO Saline BSO Saline

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SLIDE 21

C57BL/6 Treated Skin and Lung Harvested 24 hours after last treatment

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SLIDE 22

Error bars represent standard error of the mean

* * p = 0.048

2 4 6 8 10 12 14

Untreated BSO

BaP DNA Adducts per 10 9 Nucleotides

MilliQ Water Arsenite

BaP DNA Adduct Levels in Lung of C57BL/6 Mice

*

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SLIDE 23

Error bars represent standard error of the mean

* * p = 0.028

BaP DNA Adduct Levels in Skin of C57BL/6 Mouse

0.00 100.00 200.00 300.00 400.00 500.00 600.00

Untreated BSO

BaP DNA Adducts per 10

9 Nucleotides

MilliQ Water Arsenite

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SLIDE 24

1 mg/L Sodium Arsenite MilliQ Water

21 days

  • f treatment

C57BL/6 GCLM(-/-) Topical treatment of BaP – 8 daily doses

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SLIDE 25

Error bars represent standard error of the mean

Comparison of Arsenite's ability to Potentiate BaP DNA adducts in Gclm(-/-) mice versus C57BL/6 in Lung Tissue

2 4 6 8 10 12 14 16 18 20

Gclm(-/-)

BaP DNA Adducts per 10

9 Nucleotides

MilliQ Water 1 mg/L Arsenite

C57BL/6

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SLIDE 26

Comparison of Arsenite's ability to Potentiate BaP DNA adducts in Gclm(-/-) mice versus C57BL/6 in Skin Tissue

50 100 150 200 250 300 350 400 450

Gclm(-/-)

BaP DNA Adducts per 10

9 Nucleotides

MilliQ Water 1 mg/L Arsenite

C57BL/6 Error bars represent standard error of mean

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SLIDE 27

Conclusions Conclusions – – Effect of decreased GSH Effect of decreased GSH in vivo in vivo

► ►Arsenite potentiates BaP

Arsenite potentiates BaP-

  • Adduct levels in

Adduct levels in the lung but not the skin the lung but not the skin

► ►Decreased GSH increases BaP

Decreased GSH increases BaP-

  • DNA adducts

DNA adducts in the skin and not the lung in the skin and not the lung

► ►Gclm

Gclm( (-

  • /

/ -

  • )

) mice more sensitive to arsenite mice more sensitive to arsenite treatment treatment

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SLIDE 28

Phase 2: Effect of Phase 2: Effect of increased increased GSH: GSH: In vitro In vitro

► ►Characterize dose

Characterize dose-

  • response relationship

response relationship between arsenite, NAC, and BaP DNA between arsenite, NAC, and BaP DNA adducts using HEPA adducts using HEPA-

  • 1 cells

1 cells

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SLIDE 29

Cell Treatment Protocol Cell Treatment Protocol

0 hrs 12 24 36 48 Treat with Arsenite and NAC Add NAC Treat with BaP Harvest Cells

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SLIDE 30

Effect of N-acetyl cysteine on BaP DNA adducts in HEPA-1 Cells

50 100 150 200 250 300 1 5

N-Acetyl Cysteine (mM) % of DNA Adducts Compared to Baseline

(Cells treated with BaP Only)

0 uM Arsenite 0.5 uM Arsenite 1 uM Arsenite

Baseline

Error bars represent standard error of mean

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SLIDE 31

Conclusions Conclusions – – Effect of increased GSH Effect of increased GSH in vitro in vitro

► ►Augmenting GSH does not reduce BaP

Augmenting GSH does not reduce BaP-

  • DNA

DNA adducts in Hepa adducts in Hepa-

  • 1 Cells

1 Cells

  • Is the effective Glutathione transferase activity

Is the effective Glutathione transferase activity affected by treatment? affected by treatment?

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SLIDE 32

Effect of increased GSH: Effect of increased GSH: In vivo In vivo

► ►C57BL/6 Mice Treatments

C57BL/6 Mice Treatments

  • Arsenite in DW

Arsenite in DW

  • GSHEE

GSHEE

  • BaP

BaP

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SLIDE 33

2 mg/L Sodium Arsenite MilliQ Water

21 days

  • f treatment

8 daily doses administered 1 hour after topical treatment of BaP

C57BL/6

Saline GSHEE Saline GSHEE

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SLIDE 34

C57BL/6 Treated Skin and Lung Harvested 24 hours after last treatment

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SLIDE 35

Error bars represent standard error of the mean

*

2 4 6 8 10 12 14 16

Untreated GSHEE

BaP DNA Adducts per 10

9 Nucleotides

MilliQ Water 2 mg/L Arsenite

BaP DNA Adduct Levels in Lung of C57BL/6 Mouse

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SLIDE 36

Error bars represent standard error of the mean

BaP DNA Adduct Levels in Skin of C57BL/6 Mouse

0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00

Untreated GSHEE

BaP DNA Adducts per 10

9 Nucleotides

MilliQ Water 2 mg/L Arsenite

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SLIDE 37

Conclusions Conclusions – – Effect of increased GSH Effect of increased GSH in vivo in vivo

► ►GSHEE treatment does not reduce BaP

GSHEE treatment does not reduce BaP-

  • DNA

DNA adduct levels in skin or lung adduct levels in skin or lung

  • There are differences in effect on skin and lung

There are differences in effect on skin and lung

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SLIDE 38

CYP450 CYP450 CYP450 CYP450 CYP450

► ►Variation in

Variation in application to application to skin skin

► ►Skin received

Skin received highest dose highest dose

► ►Differences in

Differences in phase I and II phase I and II activity levels activity levels

Why differences in skin and lung? Why differences in skin and lung?

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SLIDE 39

Overall Conclusions Overall Conclusions

► ►Arsenite potentiates BaP

Arsenite potentiates BaP-

  • Adduct levels

Adduct levels mostly in lung mostly in lung

► ►BaP DNA adduct levels are not inversely

BaP DNA adduct levels are not inversely related to GSH concentration related to GSH concentration

  • Do treatments to enhance GSH actually increase

Do treatments to enhance GSH actually increase the activity of glutathione the activity of glutathione-

  • S

S-

  • transferase?

transferase? ► ►Is this the smoking gun of Arsenic

Is this the smoking gun of Arsenic’ ’s s carcinogenity carcinogenity??? ???

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SLIDE 40

Future Direction Future Direction

► ►Examine Arsenic effect on DNA repair IN

Examine Arsenic effect on DNA repair IN Vivo Vivo

► ►Arsenic speciation

Arsenic speciation

► ►Will longer BAP treatments lead to more

Will longer BAP treatments lead to more pronounced effects? pronounced effects?

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SLIDE 41

Acknowledgements Acknowledgements

► ►Brenda Schumann

Brenda Schumann

► ►Kathy LaDow

Kathy LaDow

► ►Mustafa Al

Mustafa Al-

  • Zoughool

Zoughool

► ►Ying Chen

Ying Chen

► ►Scott Schneider

Scott Schneider

► ►NIOSH Pilot Research Project Training

NIOSH Pilot Research Project Training Program of the University of Cincinnati ERC Program of the University of Cincinnati ERC Grant # T42/OH008432 Grant # T42/OH008432-

  • 01

01

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SLIDE 42
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SLIDE 43

Backup Slides Backup Slides

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SLIDE 44

Effect of BSO and Arsenite Treatment on GSH Levels in Hepa Effect of BSO and Arsenite Treatment on GSH Levels in Hepa-

  • 1 Cells

1 Cells

1 2 3 4 5 6 7 8 9 5 10 15 20 25 30 35 40 45 50

Time (hrs) GSH (nmol/mg protein)

1 mM BSO 1 mM BSO + 1 uM As 5 mM BSO 5 mM BSO + 1 uM As

µ µ µ µ

Error bars represent standard error of mean

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SLIDE 45

Effect of BSO Treatments on GSH Levels in Hepa Effect of BSO Treatments on GSH Levels in Hepa-

  • 1 Cells

1 Cells

Error bars represent standard error of mean

1 2 3 4 5 6 7 8 9 5 10 15 20 25 30 35 40 45 50

Time (hrs)

GSH (nmol/mg protein)

Untreated 1 mM BSO 5 mM BSO 10 mM BSO 15 mM BSO

µ µ µ µ

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SLIDE 46

Model of Arsenite Metabolism Model of Arsenite Metabolism

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SLIDE 47

Purpose Purpose

► ►Understand glutathione

Understand glutathione’ ’s role in arsenic and s role in arsenic and benzo(a)pyrene benzo(a)pyrene co co-

  • carcinogenesis

carcinogenesis

  • Are glutathione deficient persons more at risk?

Are glutathione deficient persons more at risk?

  • Do increased levels of glutathione provide

Do increased levels of glutathione provide protection? protection?

  • Can supplementation/treatment using

Can supplementation/treatment using glutathione be provided? glutathione be provided?

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SLIDE 48

2 4 6 8 10 12 14 16 18 20 5 10 15 20 25 30 35 40 45 50

Time (hrs) GSH (nmol/mg protein)

Untreated 1 mM NAC 5 mM NAC

Effect of NAC Treatments on GSH Levels in Hepa Effect of NAC Treatments on GSH Levels in Hepa-

  • 1 Cells

1 Cells

Error bars represent standard error of mean

slide-49
SLIDE 49

5 10 15 20 25 30

10 20 30 40 50

Time (hrs) GSH (nmol/mg protein)

1 mM NAC 5 mM NAC 1 mM NAC + 1 uM As 5 mM NAC + 1 uM As

Effect of NAC and Arsenite Treatments on GSH Levels in Hepa Effect of NAC and Arsenite Treatments on GSH Levels in Hepa-

  • 1 Cells

1 Cells

Error bars represent standard error of mean

slide-50
SLIDE 50
slide-51
SLIDE 51
slide-52
SLIDE 52

Study Aims Study Aims

► ►-

  • Study the effect of increased and

Study the effect of increased and decreased levels of glutathione on decreased levels of glutathione on arsenite arsenite’ ’s s impact on impact on benzo(a)pyrene benzo(a)pyrene DNA adduct levels DNA adduct levels in vivo in vivo. .

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SLIDE 53

Acknowledgements Acknowledgements

► ►Howard Shertzer, PhD

Howard Shertzer, PhD

► ►Paul Succop, PhD

Paul Succop, PhD

► ►Joseph Caruso, PhD

Joseph Caruso, PhD

► ►Tim Dalton, PhD

Tim Dalton, PhD

► ►Alvaro Puga, PhD

Alvaro Puga, PhD

► ►Craig Tomlinson, PhD

Craig Tomlinson, PhD

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SLIDE 54

Preliminary study of the Effect of Glutathione on BaP Adducts Preliminary study of the Effect of Glutathione on BaP Adducts in Hepa in Hepa-

  • 1 Cells

1 Cells

Adapted from Maier et al (2002)

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SLIDE 55

Research Questions Research Questions

► ►Do reduced GSH levels increase the risk of

Do reduced GSH levels increase the risk of arsenic arsenic cocarcinogenesis cocarcinogenesis? ?

► ►Does increased GSH reduce risk of arsenic

Does increased GSH reduce risk of arsenic cocarcinogenesis cocarcinogenesis? ?

  • Treatment / Supplementation

Treatment / Supplementation

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SLIDE 56

Error bars represent standard error of the mean

Dose Response of Arsenite on DNA Adduct levels in the Lung of C57BL/6 Mice

2 4 6 8 10 12

0 mg/L 2 mg/L

10 mg/L

BaP DNA Adducts per 10

9 Nucleotides

slide-57
SLIDE 57

9.20 9.20 7.54 7.54 299.6 299.6 272.3 272.3 Yes Yes No No 800 800 nmol nmol Vietas C57BL/6 Study Vietas C57BL/6 Study 19.93 19.93 12.41 12.41 364.6 364.6 307.1 307.1 Yes Yes No No 1000 1000 nmol nmol Evans Evans et al. ( et al. (2004) 2004) Mean Lung Adducts Mean Lung Adducts (per 10 (per 109

9

Nucleotides) Nucleotides) Mean Skin Adducts Mean Skin Adducts (per 10 (per 109

9

Nucleotides) Nucleotides) Arsenite Arsenite Total BaP Total BaP Dose Dose Study Study

Increase: 54% Increase: 19%