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

Transcription

1 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

2 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

3 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

4 Modeling Arsenic Problems Problems with animal model As not a complete carcinogen Cancer observed at cytotoxic levels Co-CarcinogenesisCarcinogenesis UV BaP

5 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

6 Effect of Arsenite on BaP Adducts in Hepa-1 1 Cells Fold change in adduct level M BaP 0.5 M BaP 0.1 M BaP 17x µm As Maier et al. (2002) Mutat. Res., 517, 101

7 Effect of Arsenite on Lung BaP Adducts in C57BL/6 Mice 50 Adducts per 10 9 Nucleotides 19.9 * As + BaP BaP Evans et al. (2004) Carcinogenesis, 25, 493 * p = 0.038

8 Effect of Arsenite on Skin BaP Adducts in C57BL/6 Mice Adducts per 10 9 Nucleotides As + BaP BaP Evans et al. (2004) Carcinogenesis

9 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

10 Potential Mechanism for BAP and As Interactions

11 Glutathione (GSH) Function Major thiol in redox homeostasis mm in all cells Conjugates with electrophiles Synthesis Six Enzymes Glutamate-cysteine ligase (GCL)

12 Glutathione Modulation GCL GCL Inhibitors Buthionine Sulfoximine (BSO) GCLM (-/-)( Increasing GSH Glutathione ethyl ester (GSH-EE) N-acetyl cysteine

13 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.

14 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

15 Cell Treatment Protocol 0 hrs Treat with Arsenite and BSO Add BSO Treat with BaP Harvest Cells

16 500 BaP DNA Adduct Levels in Hepa-1 Cells Treated with Arsenite and BSO % BaP DNA Adducts compared to Baseline (no arsenite or BSO treatment) Baseline 0 µm As 0.5 µm As 1 µm As 2 µm As Buthionine Sulfoximine (µm) Error bars represent standard error of mean

17 Effect of Arsenite on BaP DNA Adducts in Gclm(+/+) and Gclm(-/-) Mouse Embryonic Fibroblasts 400 BaP DNA Adduct Level per 10 9 Nucleotides um As um As 0 Gclm (+/+) Gclm (-/-) Error bars represent standard error of mean

18 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

19 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

20 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

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

22 BaP DNA Adducts per 10 9 Nucleotides BaP DNA Adduct Levels in Lung of C57BL/6 Mice MilliQ Water Arsenite * * 0 Untreated BSO * p = Error bars represent standard error of the mean

23 BaP DNA Adduct Levels in Skin of C57BL/6 Mouse BaP DNA Adducts per 10 9 Nucleotides MilliQ Water Arsenite 0.00 * p = Untreated BSO * Error bars represent standard error of the mean

24 C57BL/6 GCLM(-/-) 1 mg/l Sodium Arsenite 21 days of treatment MilliQ Water Topical treatment of BaP 8 daily doses

25 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 MilliQ Water 1 mg/l Arsenite 2 0 C57BL/6 Gclm(-/-) Error bars represent standard error of the mean

26 BaP DNA Adducts per 10 9 Nucleotides 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

27 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

28 Phase 2: Effect of increased GSH: In vitro Characterize dose-response relationship between arsenite, NAC, and BaP DNA adducts using HEPA-1 1 cells

29 Cell Treatment Protocol 0 hrs Treat with Arsenite and NAC Add NAC Treat with BaP Harvest Cells

30 Effect of N-acetyl cysteine on BaP DNA adducts in HEPA-1 Cells um Arsenite % of DNA Adducts Compared to Baseline (Cells treated with BaP Only) Baseline 0.5 um Arsenite 1 um Arsenite N-Acetyl Cysteine (mm) Error bars represent standard error of mean

31 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?

32 Effect of increased GSH: In vivo C57BL/6 C57BL/6 Mice Treatments Arsenite in DW GSHEE BaP

33 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

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

35 BaP DNA Adduct Levels in Lung of C57BL/6 Mouse BaP DNA Adducts per 10 9 Nucleotides MilliQ Water 2 mg/l Arsenite * 0 Untreated GSHEE Error bars represent standard error of the mean

36 BaP DNA Adduct Levels in Skin of C57BL/6 Mouse BaP DNA Adducts per 10 9 Nucleotides MilliQ Water 2 mg/l Arsenite Untreated GSHEE Error bars represent standard error of the mean

37 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

38 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

39 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???

40 Future Direction Examine Examine Arsenic effect on DNA repair IN Vivo Arsenic Arsenic speciation Will Will longer BAP treatments lead to more pronounced effects?

41 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/OH

42

43 Backup Slides

44 9 Effect of BSO and Arsenite Treatment on GSH Levels in Hepa-1 1 Cells 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) Time (hrs) Error bars represent standard error of mean

45 Effect of BSO Treatments on GSH Levels in Hepa-1 1 Cells GSH (nmol/mg protein) Untreated 1 mm µ BSO 5 mm µ BSO 10 mm µ BSO 15 mm µ BSO Time (hrs) Error bars represent standard error of mean

46 Model of Arsenite Metabolism

47 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?

48 Effect of NAC Treatments on GSH Levels in Hepa-1 1 Cells GSH (nmol/mg protein) Untreated 1 mm NAC 5 mm NAC Time (hrs) Error bars represent standard error of mean

49 Effect of NAC and Arsenite Treatments on GSH Levels in Hepa-1 1 Cells mm NAC 5 mm NAC 1 mm NAC + 1 um As 5 mm NAC + 1 um As GSH (nmol/mg protein) Time (hrs) Error bars represent standard error of mean

50

51

52 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.

53 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

54 Preliminary study of the Effect of Glutathione on BaP Adducts in Hepa-1 1 Cells Adapted from Maier et al (2002)

55 Research Questions Do reduced GSH levels increase the risk of arsenic cocarcinogenesis? Does Does increased GSH reduce risk of arsenic cocarcinogenesis? Treatment / Supplementation

56 Dose Response of Arsenite on DNA Adduct levels in the Lung of C57BL/6 Mice 12 BaP DNA Adducts per 10 9 Nucleotides mg/l 2 mg/l 10 mg/l Error bars represent standard error of the mean

57 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 Increase: 54% Vietas C57BL/6 Study 800 nmol Yes No Increase: 19%