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 Vietas 1 Department of Environmental Health, 1 Department of Environmental Health, 2 United States Air University of Cincinnati, 2 United States Air University of Cincinnati, Force Force
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 observed increased cancer risk observed increased cancer risk
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
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
Benzo(a)pyrene Benzo(a)pyrene ► Common component Common component ► of PAH mixtures: coal of 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
Effect of Arsenite on BaP Adducts in Effect of Arsenite on BaP Adducts in Hepa- -1 Cells 1 Cells Hepa Fold change in adduct level 1.0 M BaP 100 0.5 M BaP 10 0.1 M BaP 17x 1 0 5 10 µ M As Maier et al . (2002) Mutat. Res., 517, 101
Effect of Arsenite on Lung BaP Adducts in C57BL/6 Mice 50 9 Nucleotides Adducts per 10 19.9 * 12.7 0 As + BaP BaP * p = 0.038 Evans et al. (2004) Carcinogenesis, 25 , 493
Effect of Arsenite on Skin BaP Adducts in C57BL/6 Mice 500 450 400 Adducts per 10 9 Nucleotides 350 364.1 300 307.1 250 200 150 100 50 0 As + BaP BaP Evans et al. (2004) Carcinogenesis
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
Potential Mechanism for BAP and As Potential Mechanism for BAP and As Interactions Interactions
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)
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
Hypothesis Hypothesis Changes in intracellular glutathione Changes in intracellular glutathione levels are inversely inversely related to related to levels are arsenite’ ’s s effect on effect on benzo(a)pyrene benzo(a)pyrene arsenite DNA adduct levels in vitro and in in vitro and in DNA adduct levels vivo. . vivo
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- -1 cells 1 cells adducts using HEPA ► Observe the effect of endogenous reduction ► Observe the effect of endogenous reduction of glutathione levels using Gclm Gclm( (- -/ / - -) ) MEFs MEFs of glutathione levels using
Cell Treatment Protocol Cell Treatment Protocol 0 hrs 12 24 36 48 Treat with Harvest Arsenite Cells Treat with Add BSO BaP and BSO
BaP DNA Adduct Levels in Hepa-1 Cells Treated with Arsenite and BSO 500 0 µM As 450 % BaP DNA Adducts compared to Baseline 0.5 µM As 1 µM As 400 2 µM As (no arsenite or BSO treatment) 350 300 250 200 150 100 Baseline 50 0 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 9 Nucleotides 0 uM As 350 0.125 uM As 300 BaP DNA Adduct Level per 10 250 200 150 100 50 0 Gclm (+/+) Gclm (-/-) Error bars represent standard error of mean
Conclusions – – Conclusions Effect of decreased GSH in vitro in vitro Effect of decreased GSH ► 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’ ’s sensitivity to arsenite s sensitivity to arsenite the cell
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
C57BL/6 21 days 2 mg/L MilliQ Water of treatment Sodium Arsenite 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 Adduct Levels in Lung of C57BL/6 Mice 14 MilliQ Water * * Arsenite 12 BaP DNA Adducts per 10 9 Nucleotides 10 8 6 4 2 0 Untreated BSO * p = 0.048 Error bars represent standard error of the mean
BaP DNA Adduct Levels in Skin of C57BL/6 Mouse 600.00 MilliQ Water Arsenite 9 Nucleotides 500.00 400.00 BaP DNA Adducts per 10 300.00 200.00 100.00 0.00 * Untreated BSO * p = 0.028 Error bars represent standard error of the mean
C57BL/6 GCLM(-/-) 1 mg/L 21 days MilliQ Water Sodium Arsenite of treatment 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 20 MilliQ Water 18 1 mg/L Arsenite 9 Nucleotides 16 14 BaP DNA Adducts per 10 12 10 8 6 4 2 0 C57BL/6 Gclm(-/-) 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 Skin Tissue 450 MilliQ Water 400 1 mg/L Arsenite 9 Nucleotides 350 300 BaP DNA Adducts per 10 250 200 150 100 50 0 C57BL/6 Gclm(-/-) Error bars represent standard error of mean
Conclusions – – Conclusions Effect of decreased GSH in vivo in vivo Effect of decreased GSH ► 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
Phase 2: Effect of increased increased GSH: GSH: Phase 2: Effect of 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- -1 cells 1 cells adducts using HEPA
Cell Treatment Protocol Cell Treatment Protocol 0 hrs 12 24 36 48 Treat with Harvest Arsenite Cells Treat with Add NAC and NAC BaP
Effect of N-acetyl cysteine on BaP DNA adducts in HEPA-1 Cells 300 0 uM Arsenite % of DNA Adducts Compared to Baseline 0.5 uM Arsenite 250 1 uM Arsenite (Cells treated with BaP Only) 200 150 100 Baseline 50 0 0 1 5 N-Acetyl Cysteine (mM) Error bars represent standard error of mean
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