Co-exposure of Arsenite and Benzo(a)pyrene: : Effect of glutathione on DNA adduct levels Glenn Talaska 1 Jay Vietas 1,2 1 Department of Environmental Health, University of Cincinnati, 2 United States Air Force
Arsenic and Cancer Medicinal Medicinal Uses and Occupational Exposures Higher Higher dose chronic studies (>100 ug/l) Tseng et al.. (1968) in Taiwan Confirmation in other studies Chile, Chile, China, Bangladesh Lower Lower dose studies (<100 ug/l) have not observed increased cancer risk
Occupational Epidemiology Occupational Exposures Smelters, pesticides, fossil fuel combustion Strong Strong relation between cancer risk and: Length of exposure Amount of exposure Interaction effect of smoking and arsenic
Modeling Arsenic Problems Problems with animal model As not a complete carcinogen Cancer observed at cytotoxic levels Co-CarcinogenesisCarcinogenesis UV BaP
Benzo(a)pyrene Common component of PAH mixtures: coal tar, tobacco smoke Used to model entire family of PAHs Exposure results in formation of stable DNA adducts Measurable with sensitive assay
Effect of Arsenite on BaP Adducts in Hepa-1 1 Cells Fold change in adduct level 100 10 1 1.0 M BaP 0.5 M BaP 0.1 M BaP 17x 0 5 10 µm As Maier et al. (2002) Mutat. Res., 517, 101
Effect of Arsenite on Lung BaP Adducts in C57BL/6 Mice 50 Adducts per 10 9 Nucleotides 19.9 * 12.7 0 As + BaP BaP Evans et al. (2004) Carcinogenesis, 25, 493 * p = 0.038
Effect of Arsenite on Skin BaP Adducts in C57BL/6 Mice 500 450 Adducts per 10 9 Nucleotides 400 350 300 250 200 150 100 364.1 307.1 50 0 As + BaP BaP Evans et al. (2004) Carcinogenesis
Common Link Arsenite Arsenite Metabolism Series of steps involving reduction and methylation Requires glutathione as a cofactor Benzo(a)pyrene Detoxification Glutathione conjugation Common Common biological link: Glutathione
Potential Mechanism for BAP and As Interactions
Glutathione (GSH) Function Major thiol in redox homeostasis 1-1010 mm in all cells Conjugates with electrophiles Synthesis Six Enzymes Glutamate-cysteine ligase (GCL)
Glutathione Modulation GCL GCL Inhibitors Buthionine Sulfoximine (BSO) GCLM (-/-)( Increasing GSH Glutathione ethyl ester (GSH-EE) N-acetyl cysteine
Hypothesis Changes in intracellular glutathione levels are inversely related to arsenite s effect on benzo(a)pyrene DNA adduct levels in vitro and in vivo.
Phase 1: Effect of decreased GSH In vitro Characterize dose-response relationship between arsenite, BSO, and BaP DNA adducts using HEPA-1 1 cells Observe Observe the effect of endogenous reduction of glutathione levels using Gclm(-/-) MEFs
Cell Treatment Protocol 0 hrs 12 24 36 48 Treat with Arsenite and BSO Add BSO Treat with BaP Harvest Cells
500 BaP DNA Adduct Levels in Hepa-1 Cells Treated with Arsenite and BSO % BaP DNA Adducts compared to Baseline (no arsenite or BSO treatment) 450 400 350 300 250 200 150 100 50 0 Baseline 0 µm As 0.5 µm As 1 µm As 2 µm As 0 1 5 10 15 Buthionine Sulfoximine (µm) Error bars represent standard error of mean
Effect of Arsenite on BaP DNA Adducts in Gclm(+/+) and Gclm(-/-) Mouse Embryonic Fibroblasts 400 BaP DNA Adduct Level per 10 9 Nucleotides 350 300 250 200 150 100 50 0 um As 0.125 um As 0 Gclm (+/+) Gclm (-/-) Error bars represent standard error of mean
Conclusions Effect of decreased GSH in vitro Arsenite Arsenite potentiates BaP DNA adduct levels As GSH decreases, BaP DNA adduct levels increase Decreased GSH levels appear to increase the cell s s sensitivity to arsenite
Phase 1: Effect of decreased GSH: In vivo C57BL/6 C57BL/6 Mice Treatments Arsenite in DW BSO BaP Wild Wild Type and Glcm (-/-) Arsenite in DW BaP
C57BL/6 2 mg/l Sodium Arsenite 21 days of treatment MilliQ Water BSO Saline BSO Saline 8 daily doses administered 1 hour after topical treatment of BaP
C57BL/6 24 hours after last treatment Treated Skin and Lung Harvested
BaP DNA Adducts per 10 9 Nucleotides 14 12 10 8 6 4 2 BaP DNA Adduct Levels in Lung of C57BL/6 Mice MilliQ Water Arsenite * * 0 Untreated BSO * p = 0.048 Error bars represent standard error of the mean
BaP DNA Adduct Levels in Skin of C57BL/6 Mouse BaP DNA Adducts per 10 9 Nucleotides 600.00 500.00 400.00 300.00 200.00 100.00 MilliQ Water Arsenite 0.00 * p = 0.028 Untreated BSO * Error bars represent standard error of the mean
C57BL/6 GCLM(-/-) 1 mg/l Sodium Arsenite 21 days of treatment MilliQ Water Topical treatment of BaP 8 daily doses
Comparison of Arsenite's ability to Potentiate BaP DNA adducts in Gclm(-/-) mice versus C57BL/6 in Lung Tissue BaP DNA Adducts per 10 9 Nucleotides 20 18 16 14 12 10 8 6 4 MilliQ Water 1 mg/l Arsenite 2 0 C57BL/6 Gclm(-/-) Error bars represent standard error of the mean
BaP DNA Adducts per 10 9 Nucleotides 450 400 350 300 250 200 150 100 Comparison of Arsenite's ability to Potentiate BaP DNA adducts in Gclm(-/-) mice versus C57BL/6 in Skin Tissue MilliQ Water 1 mg/l Arsenite 50 0 C57BL/6 Gclm(-/-) Error bars represent standard error of mean
Conclusions Effect of decreased GSH in vivo Arsenite Arsenite potentiates BaP-Adduct levels in the lung but not the skin Decreased GSH increases BaP-DNA adducts in the skin and not the lung Gclm(-/-) mice more sensitive to arsenite treatment
Phase 2: Effect of increased GSH: In vitro Characterize dose-response relationship between arsenite, NAC, and BaP DNA adducts using HEPA-1 1 cells
Cell Treatment Protocol 0 hrs 12 24 36 48 Treat with Arsenite and NAC Add NAC Treat with BaP Harvest Cells
Effect of N-acetyl cysteine on BaP DNA adducts in HEPA-1 Cells 300 0 um Arsenite % of DNA Adducts Compared to Baseline (Cells treated with BaP Only) 250 200 150 100 50 Baseline 0.5 um Arsenite 1 um Arsenite 0 0 1 5 N-Acetyl Cysteine (mm) Error bars represent standard error of mean
Conclusions Effect of increased GSH in vitro Augmenting GSH does not reduce BaP-DNA adducts in Hepa-1 1 Cells Is the effective Glutathione transferase activity affected by treatment?
Effect of increased GSH: In vivo C57BL/6 C57BL/6 Mice Treatments Arsenite in DW GSHEE BaP
C57BL/6 2 mg/l Sodium Arsenite 21 days of treatment MilliQ Water GSHEE Saline GSHEE Saline 8 daily doses administered 1 hour after topical treatment of BaP
C57BL/6 24 hours after last treatment Treated Skin and Lung Harvested
BaP DNA Adduct Levels in Lung of C57BL/6 Mouse BaP DNA Adducts per 10 9 Nucleotides 16 14 12 10 8 6 4 2 MilliQ Water 2 mg/l Arsenite * 0 Untreated GSHEE Error bars represent standard error of the mean
BaP DNA Adduct Levels in Skin of C57BL/6 Mouse 450.00 BaP DNA Adducts per 10 9 Nucleotides 400.00 350.00 300.00 250.00 200.00 150.00 100.00 MilliQ Water 2 mg/l Arsenite 50.00 0.00 Untreated GSHEE Error bars represent standard error of the mean
Conclusions Effect of increased GSH in vivo GSHEE GSHEE treatment does not reduce BaP-DNA adduct levels in skin or lung There are differences in effect on skin and lung
Why differences in skin and lung? Variation Variation in application to skin Skin Skin received highest dose Differences in phase I and II activity levels CYP450 CYP450 CYP450 CYP450 CYP450
Overall Conclusions Arsenite Arsenite potentiates BaP-Adduct levels mostly in lung BaP BaP DNA adduct levels are not inversely related to GSH concentration Do treatments to enhance GSH actually increase the activity of glutathione-s-transferase? transferase? Is this the smoking gun of Arsenic s carcinogenity???
Future Direction Examine Examine Arsenic effect on DNA repair IN Vivo Arsenic Arsenic speciation Will Will longer BAP treatments lead to more pronounced effects?
Acknowledgements Brenda Brenda Schumann Kathy Kathy LaDow Mustafa Mustafa Al-Zoughool Ying Ying Chen Scott Scott Schneider NIOSH NIOSH Pilot Research Project Training Program of the University of Cincinnati ERC Grant #T42/OH008432-01 01
Backup Slides
9 Effect of BSO and Arsenite Treatment on GSH Levels in Hepa-1 1 Cells 8 7 1 mm µ BSO 1 mm µ BSO + 1 um As 5 mm µ BSO 5 mm µ BSO + 1 um As GSH (nmol/mg protein) 6 5 4 3 2 1 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Error bars represent standard error of mean
Effect of BSO Treatments on GSH Levels in Hepa-1 1 Cells GSH (nmol/mg protein) 9 8 7 6 5 4 3 2 Untreated 1 mm µ BSO 5 mm µ BSO 10 mm µ BSO 15 mm µ BSO 1 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Error bars represent standard error of mean
Model of Arsenite Metabolism
Purpose Understand glutathione s s role in arsenic and benzo(a)pyrene co-carcinogenesis carcinogenesis Are glutathione deficient persons more at risk? Do increased levels of glutathione provide protection? Can supplementation/treatment using glutathione be provided?
Effect of NAC Treatments on GSH Levels in Hepa-1 1 Cells 20 18 16 GSH (nmol/mg protein) 14 12 10 8 6 4 Untreated 1 mm NAC 5 mm NAC 2 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Error bars represent standard error of mean
Effect of NAC and Arsenite Treatments on GSH Levels in Hepa-1 1 Cells 30 25 1 mm NAC 5 mm NAC 1 mm NAC + 1 um As 5 mm NAC + 1 um As GSH (nmol/mg protein) 20 15 10 5 0 0 10 20 30 40 50 Time (hrs) Error bars represent standard error of mean
Study Aims - Study the effect of increased and decreased levels of glutathione on arsenite s impact on benzo(a)pyrene DNA adduct levels in vivo.
Acknowledgements Howard Howard Shertzer, PhD Paul Paul Succop, PhD Joseph Joseph Caruso, PhD Tim Tim Dalton, PhD Alvaro Alvaro Puga, PhD Craig Craig Tomlinson, PhD
Preliminary study of the Effect of Glutathione on BaP Adducts in Hepa-1 1 Cells Adapted from Maier et al (2002)
Research Questions Do reduced GSH levels increase the risk of arsenic cocarcinogenesis? Does Does increased GSH reduce risk of arsenic cocarcinogenesis? Treatment / Supplementation
Dose Response of Arsenite on DNA Adduct levels in the Lung of C57BL/6 Mice 12 BaP DNA Adducts per 10 9 Nucleotides 10 8 6 4 2 0 0 mg/l 2 mg/l 10 mg/l Error bars represent standard error of the mean
Study Total BaP Dose Arsenite Mean Skin Adducts (per 10 9 Nucleotides) Mean Lung Adducts (per 10 9 Nucleotides) Evans et al. (2004) ( 1000 nmol Yes No 364.6 307.1 19.93 12.41 Increase: 54% Vietas C57BL/6 Study 800 nmol Yes No 299.6 272.3 9.20 7.54 Increase: 19%