Analyzing Genomic Dose-Response Information to Inform Key Events in a Mode of Action for Carcinogenicity: An Example with Arsenic Analyzing Genomic Dose-Response Information to Inform Key Events in a Mode of Action for Carcinogenicity: An Example with Arsenic Robinan Gentry, PhD, DABT ENVIRON International Corporation RASS Monthly Telecon June 9, 2010

2 Background  Evidence from epidemiological studies has indicates that exposure to high concentrations of inorganic arsenic in air or in drinking water is associated with an increased risk of cancer in human populations.  Chronic human exposures to high arsenic concentrations are associated with lung, skin, and bladder cancer.

3 Background  One feature of the carcinogenicity of inorganic arsenic is the observation that human exposures have been associated with increases in skin, lung, and internal cancers, but inorganic arsenic has not historically caused tumors in standard laboratory animal studies.

4 Recent Issues  Whether there is epidemiological evidence of a “threshold” for the carcinogenic effects of inorganic arsenic, or at least of a highly nonlinear dose- response.  While arsenic has typically not produced tumors in standard animal bioassays, it has not been determined if the lack of an animal model is due to a difference in kinetics or a difference in response of cells to arsenite.  Species metabolic differences in the production of methylarsonous acid (MMAIII) have been speculated to be the contributing factor.

5 Modes of Action Several modes of action for the carcinogenicity of inorganic arsenic have been proposed. Several modes of action for the carcinogenicity of inorganic arsenic have been proposed. Reactive oxygen species protective response (Ding et al. 2005; Kligerman and Tennant 2007) Reactive oxygen species protective response (Ding et al. 2005; Kligerman and Tennant 2007) Enhancement/suppression of DNA repair activity (Kitchin 2001; Schoen et al. 2004; Snow et al. 2005; Klein et al. 2007) Enhancement/suppression of DNA repair activity (Kitchin 2001; Schoen et al. 2004; Snow et al. 2005; Klein et al. 2007) Delays in cell cycle controls (Clewell et al. 1999; Klein et al. 2007) Delays in cell cycle controls (Clewell et al. 1999; Klein et al. 2007) Activation of mitogenic responses (MAPK, VEGF, etc. ) (Tanaka- Kagawa et al. 2003) Activation of mitogenic responses (MAPK, VEGF, etc. ) (Tanaka- Kagawa et al. 2003) Apoptosis with restorative hyperplasia (Snow e tal. 2005; Klein et al. 2007) Apoptosis with restorative hyperplasia (Snow e tal. 2005; Klein et al. 2007) Few have sufficient data to support any strong conclusions. Few have sufficient data to support any strong conclusions.

6 Modes of action Common element is that most proposed MOAs are likely a sequence of dose-dependent transitions in gene or protein expression. Common element is that most proposed MOAs are likely a sequence of dose-dependent transitions in gene or protein expression. Nel et al. (2006) proposed a biological cascade of adaptive to proliferative to apoptotic responses for environmental particulates. Nel et al. (2006) proposed a biological cascade of adaptive to proliferative to apoptotic responses for environmental particulates. Represented an alternative approach at the time, but the dose-response for these interactions had not been well- characterized. Represented an alternative approach at the time, but the dose-response for these interactions had not been well- characterized.

7 Project Plan Conduct a comprehensive review of the literature focusing on gene or protein expression changes. Conduct a comprehensive review of the literature focusing on gene or protein expression changes. Organize the results as it related to arsenic concentration. Organize the results as it related to arsenic concentration. Do the available data support a cascade of biological responses progressing from adaptive to proliferative responses? Do the available data support a cascade of biological responses progressing from adaptive to proliferative responses?

8 Approach  Conducted a comprehensive literature search concentrating on the identification of quantitative gene/protein expression changes from both in vitro and in vivo studies  All citations and abstract downloaded into an endnote database to facilitate identification for review.  Quantitative dose-response information was entered into an excel database.  Organized by compound, exposure dose/concentration, species, tissue, and cell type

9 Approach  Changes in gene or protein expression were grouped by functional category (i.e., oxidative stress, DNA repair).  For each gene or protein evaluated, the lowest concentration associated with a significant increase or decrease in expression was identified.  The “matrix” was then evaluated for comparison of changes by functional category and dose.

10 Database Structure

11 SUMMARY OF THE INFORMATION IDENTIFIED IN THE LITERATURE DESCRIBING CHANGES IN GENE/PROTEIN EXPRESSION ASSOCIATED WITH A SPECIFIC CONCENTRATION OF INORGANIC ARSENIC Type of Data Total Number Total number of studies providing information on changes in specific genes and/or proteins and an associated concentration of arsenic 1160 Total number of changes in specific genes or proteins identified in vitro with an associated concentration of arsenic 700 From tumor-derived cell lines From tumor-derived cell lines230 From primary cell lines (either normal or immortalized) From primary cell lines (either normal or immortalized)470 Total changes in specific genes identified in vivo 427 From tumor-derived cell lines From tumor-derived cell lines44 From primary cell lines (either normal or immortalized) From primary cell lines (either normal or immortalized)383 1 Note that each study identified may provide changes in expression for multiple genes or proteins (e.g., increases in both VEGF and ERK ).

12 Analyses Conducted  In vivo studies  In vitro studies  Primary cells  Immortalized cells  Tumor-derived cells

13 In Vivo Studies Approximately 400 data points were identified after administration of inorganic arsenic.  Majority of the studies did not evaluate the critical target organs of concern in humans ( i.e., lung, skin or bladder).  Focused on changes in selected genes or proteins in mouse liver following chronic or in utero administration.  High water concentrations (45 ppm or greater) or injection studies (2 mg/kg/day or greater).

14 In Vivo Studies  Mouse liver gene changes related to cell cycle regulation, growth factors and hormone receptors, apoptosis and stress at high concentrations.  Simeonova et al. (2000, 2002) – mouse bladder; focused on proteins critical to EGFR-ERK pathway.  Only a single concentration providing limited information.

15 In Vitro – Primary Cells  Evaluation included in vitro studies in primary cell lines  Genes/proteins were stratified by the lowest concentration that produced a change in gene/protein levels  Genes/proteins that produced changes at identical concentrations were grouped  Each group of genes/proteins were evaluated

16 DOSE-RESPONSE FOR THE IN VITRO EFFECTS OF ARSENIC IN PRIMARY CELLS

17 In Vitro – Immortalized Cells  Same evaluation process used for primary cells in that genes/proteins were plotted by the lowest concentration that produced a change in gene/protein levels  Genes/proteins that produced changes at identical concentrations were grouped  Each group of genes/proteins were evaluated

18 DOSE-RESPONSE FOR THE IN VITRO EFFECTS OF ARSENIC IN IMMORTALIZED CELLS

19 Immortalized vs. Primary  Based on the available data, immortalized cells seem to respond similarly for some endpoints; however, there is limited information in the low-concentration region.  Some genes associated with proliferation ( e.g., VEGF) demonstrate increased expression at lower concentrations in primary cells (1 µM versus 50 µM) following arsenic exposure than in immortalized cells.  Genes associated with oxidative stress, proliferation and apoptosis exhibited increased expression following acute exposure, but decreased expression following chronic exposure in immortalized cells. Similar studies were not conducted with primary cells.  Immortalized cells provide impact of duration of exposure.

20 Immortalized vs. Primary  General consistency between dose-response patterns to arsenite for primary and immortalized cells.  Important because information on the impact of duration of exposure is onlyl available for immortalized cells (Vogt and Rossman 2001; Hu et al. 2002).  Increase in expression following acute exposure (less than 72 hours); decrease following chronic exposure (> 7 days).

21 In Vitro – Tumor-Derived Cells  Majority of the information was at higher concentrations (> 50 µM).  Depending upon the cancer type, various cell lines may be sensitive to arsenic-induced apoptosis (chemotherapy). E.g., apoptotic genes (e.g., JNK) may be activated at lower concentrations than in primary cells; in the ST486 B-lymphoma cell line, 80% apoptosis was observed at 5µM (Muscarella and Bloom 2002).  Some genes are functionally inactive in cancerous cells. In human T- cell leukemia virus type-1 (HTLV-1), p53, critical to cell cycle control, is functionally inactive.  Some genes are over-expressed. In cervical cancer cells, the over- expression of Bcl-2 contributes to the chemotherapeutic properties of arsenic.

22 Conclusions  Concentration-Response information from primary cells provides clear evidence of concentration dependence on expression  Low concentrations (below 0.1 µM) – adaptive state  0.1 to 10 µM – increased expression associated cell cycle control.  ~5 µM or greater – evidence of cytotoxicity  ~1 to 10 µM – increase in genes or proteins associated with proliferative signaling.  >10 µM – cell cycle stasis and apoptotic responses

23 Conclusions  Consistency in:  results across cell lines (primary vs. immortalized)  patterns across primary cell lines  Across cancer and noncancer responses  Consistency important for application of results in development of a mode of action

24 Proposed Mode of Action  The available in vitro data in primary cells provide clear evidence of dose-dependence of the effects of arsenic compounds on various genes or proteins from concentrations of to up to 1000 µM.  The available evidence for genomic effects in the low- concentration region, combined with information on the metabolism and protein binding, supports a mode of action for inorganic arsenic carcinogenicity involving specific direct interactions with critical proteins overlaid against a background of chemical stress.

25 Proposed Mode of Action  Key Events appear to include:  DNA repair inhibition under conditions of oxdiative stress, inflammation, and proliferative signaling, leading to a situation in which the cell is no longer able to maintain the integrity of its DNA before division

26 RELATIONSHIPS BETWEEN DIFFERENT ELEMENTS IN A MODE OF ACTION FOR THE CARCINOGENICITY OF INORGANIC ARSENIC

27 Data Gaps  Limited parallel information across species and tissue is available to conduct the cross-species comparisons needed for the human risk assessment.  Additional studies are needed in the appropriate species following a chronic duration in which the tissues are brought to steady state, such as subchronic drinking water or inhalation studies. Acute studies provide limited information due to the transient nature of some gene expressions.

28 Ongoing Research  An in vivo study in the mouse has been completed to evaluate changes in gene expression in the mouse bladder following sub-chronic exposure to arsenic.  Genomic analyses are completed in the mouse and are ongoing for human bladder epithelial cells exposed in vitro at concentrations equivalent to those achieved in the in vivo study.  The results of these studies are anticipated to be components in a nonlinear bladder cancer risk model.

29 ACKNOWLEDGEMENTS ENVIRON InternationalThe Hamner Annette ShippHarvey Clewell Dexter Sullivan Tracy McDonald Yager Environmental Janice Yager Funding EPRI