Analyzing Genomic Dose-Analyzing Genomic Dose-Response InformationResponse Information to to

Inform Key Events in a Mode of Inform Key Events in a Mode of Action for Carcinogenicity: Action for Carcinogenicity:

An Example with ArsenicAn Example with Arsenic    Robinan Gentry, PhD, DABTRobinan Gentry, PhD, DABT

ENVIRON International ENVIRON International CorporationCorporation

RASS Monthly TeleconRASS Monthly TeleconJune 9, 2010June 9, 2010

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BackgroundBackground

Evidence from epidemiological studies Evidence from epidemiological studies has indicates that exposure to high has indicates that exposure to high concentrations of inorganic arsenic in concentrations of inorganic arsenic in air or in drinking water is associated air or in drinking water is associated with an increased risk of cancer in with an increased risk of cancer in human populations. human populations.

Chronic human exposures to high Chronic human exposures to high arsenic concentrations are associated arsenic concentrations are associated with lung, skin, and bladder cancer.with lung, skin, and bladder cancer.

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BackgroundBackground

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

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Recent IssuesRecent Issues Whether there is epidemiological evidence of a Whether there is epidemiological evidence of a

“threshold” for the carcinogenic effects of “threshold” for the carcinogenic effects of inorganic arsenic, or at least of a highly nonlinear inorganic arsenic, or at least of a highly nonlinear dose-response.dose-response.

While arsenic has typically not produced tumors While arsenic has typically not produced tumors in standard animal bioassays, it has not been in standard animal bioassays, it has not been determined if the lack of an animal model is due determined if the lack of an animal model is due to a difference in kinetics or a difference in to a difference in kinetics or a difference in response of cells to arsenite.response of cells to arsenite.

Species metabolic differences in the production of Species metabolic differences in the production of methylarsonous acid (MMAIII) have been methylarsonous acid (MMAIII) have been speculated to be the contributing factor.speculated to be the contributing factor.

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Modes of ActionModes of Action Several modes of action for the carcinogenicity of Several modes of action for the carcinogenicity of

inorganic arsenic have been proposed.inorganic arsenic have been proposed. Reactive oxygen species protective response (Ding et al. Reactive oxygen species protective response (Ding et al.

2005; Kligerman and Tennant 2007)2005; Kligerman and Tennant 2007) Enhancement/suppression of DNA repair activity (Kitchin Enhancement/suppression of DNA repair activity (Kitchin

2001; Schoen et al. 2004; Snow et al. 2005; Klein et al. 2007)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. Delays in cell cycle controls (Clewell et al. 1999; Klein et al.

2007)2007) Activation of mitogenic responses (MAPK, VEGF, etc. ) Activation of mitogenic responses (MAPK, VEGF, etc. )

(Tanaka-Kagawa et al. 2003)(Tanaka-Kagawa et al. 2003) Apoptosis with restorative hyperplasia (Snow e tal. 2005; Apoptosis with restorative hyperplasia (Snow e tal. 2005;

Klein et al. 2007)Klein et al. 2007) Few have sufficient data to support any strong Few have sufficient data to support any strong

conclusions.conclusions.

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Modes of actionModes of action

Common element is that most proposed MOAs Common element is that most proposed MOAs are likely a sequence of dose-dependent are likely a sequence of dose-dependent transitions in gene or protein expression.transitions in gene or protein expression.

Nel et al. (2006) proposed a biological Nel et al. (2006) proposed a biological cascade of adaptive to proliferative to cascade of adaptive to proliferative to apoptotic responses for environmental apoptotic responses for environmental particulates.particulates.

Represented an alternative approach at the Represented an alternative approach at the time, but the dose-response for these time, but the dose-response for these interactions had not been well-characterized.interactions had not been well-characterized.

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Project PlanProject Plan

Conduct a comprehensive review of Conduct a comprehensive review of the literature focusing on gene or the literature focusing on gene or protein expression changes.protein expression changes.

Organize the results as it related to Organize the results as it related to arsenic concentration.arsenic concentration.

Do the available data support a Do the available data support a cascade of biological responses cascade of biological responses progressing from adaptive to progressing from adaptive to proliferative responses?proliferative responses?

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ApproachApproach Conducted a comprehensive literature search Conducted a comprehensive literature search

concentrating on the identification of quantitative concentrating on the identification of quantitative gene/protein expression changes from both gene/protein expression changes from both in in vitrovitro and and in vivoin vivo studies studies

All citations and abstract downloaded into an All citations and abstract downloaded into an endnote database to facilitate identification for endnote database to facilitate identification for review.review.

Quantitative dose-response information was Quantitative dose-response information was entered into an excel database.entered into an excel database.

Organized by compound, exposure Organized by compound, exposure dose/concentration, species, tissue, and cell typedose/concentration, species, tissue, and cell type

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ApproachApproach

Changes in gene or protein expression were Changes in gene or protein expression were grouped by functional category (i.e., oxidative grouped by functional category (i.e., oxidative stress, DNA repair).stress, DNA repair).

For each gene or protein evaluated, the lowest For each gene or protein evaluated, the lowest concentration associated with a significant concentration associated with a significant increase or decrease in expression was identified.increase or decrease in expression was identified.

The “matrix” was then evaluated for comparison The “matrix” was then evaluated for comparison of changes by functional category and dose.of changes by functional category and dose.

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Database StructureDatabase StructureGene/ProteinAbbreviation

Gene/ProteinName

ArsenicSpecies

TargetTissue

Cell-lineCancerous/

NormalDose

In Vitro /In Vivo

Response CommentEndnoteNumber

JNK1/2 c-jun N- terminal kinase -1/2

sodium arsenite

TRL 1215 normal 200 µM In vitro

phosporylated JNK1/2 significantly decreased in transformed cells compared with control cells

2174

JNK1/2 c-jun N- terminal kinase -1/2

sodium arsenite

TRL 1215 normal 300 µM In vitro

phosporylated JNK1/2 significantly decreased in transformed cells compared with control cells

2174

JNK1/2 c-jun N- terminal kinase -1/2

sodium arsenite

TRL 1215 normal 500 µM In vitro

phosporylated JNK1/2 significantly decreased in transformed cells compared with control cells

2174

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SUMMARY OF THE INFORMATION IDENTIFIED IN THE SUMMARY OF THE INFORMATION IDENTIFIED IN THE LITERATURE DESCRIBING CHANGES IN GENE/PROTEIN LITERATURE DESCRIBING CHANGES IN GENE/PROTEIN

EXPRESSION ASSOCIATED WITH A SPECIFIC EXPRESSION ASSOCIATED WITH A SPECIFIC CONCENTRATION OF INORGANIC ARSENICCONCENTRATION OF INORGANIC ARSENIC

Type of DataType of Data Total Total NumberNumber

Total number of studies providing information on changes in specific genes

and/or proteins and an associated concentration of arsenic1 160160

Total number of changes in specific genes or proteins identified in Total number of changes in specific genes or proteins identified in vitro with an associated concentration of arsenic vitro with an associated concentration of arsenic

700 700

From tumor-derived cell lines From tumor-derived cell lines 230 230 From primary cell lines (either normal or immortalized) From primary cell lines (either normal or immortalized) 470 470 Total changes in specific genes identified in vivo Total changes in specific genes identified in vivo 427427 From tumor-derived cell lines From tumor-derived cell lines 44 44 From primary cell lines (either normal or immortalized) From primary cell lines (either normal or immortalized) 383 383

1Note that each study identified may provide changes in expression for multiple genes or proteins (e.g., increases in both VEGF and ERK).

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Analyses ConductedAnalyses Conducted

In vivoIn vivo studies studies

In vitroIn vitro studies studies

Primary cellsPrimary cells

Immortalized cellsImmortalized cells

Tumor-derived cellsTumor-derived cells

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In VivoIn Vivo Studies StudiesApproximately 400 data points were identified after Approximately 400 data points were identified after

administration of inorganic arsenic.administration of inorganic arsenic. Majority of the studies did not evaluate the critical Majority of the studies did not evaluate the critical

target organs of concern in humans (target organs of concern in humans (i.e.i.e., lung, skin or , lung, skin or bladder).bladder).

Focused on changes in selected genes or proteins in Focused on changes in selected genes or proteins in mouse liver following chronic or in utero mouse liver following chronic or in utero administration.administration.

High water concentrations (45 ppm or greater) or High water concentrations (45 ppm or greater) or injection studies (2 mg/kg/day or greater).injection studies (2 mg/kg/day or greater).

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In VivoIn Vivo Studies Studies Mouse liver gene changes related to Mouse liver gene changes related to

cell cycle regulation, growth factors cell cycle regulation, growth factors and hormone receptors, apoptosis and and hormone receptors, apoptosis and stress at high concentrations.stress at high concentrations.

Simeonova et al. (2000, 2002) – mouse Simeonova et al. (2000, 2002) – mouse bladder; focused on proteins critical to bladder; focused on proteins critical to EGFR-ERK pathway.EGFR-ERK pathway.

Only a single concentration providing Only a single concentration providing limited information.limited information.

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In Vitro In Vitro – Primary Cells– Primary Cells Evaluation included Evaluation included in vitroin vitro studies in primary cell studies in primary cell

lineslines

Genes/proteins were stratified by the lowest Genes/proteins were stratified by the lowest concentration that produced a change in concentration that produced a change in gene/protein levelsgene/protein levels

Genes/proteins that produced changes at Genes/proteins that produced changes at identical concentrations were groupedidentical concentrations were grouped

Each group of genes/proteins were evaluatedEach group of genes/proteins were evaluated

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

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In VitroIn Vitro – Immortalized Cells – Immortalized Cells

Same evaluation process used for primary cells in Same evaluation process used for primary cells in that genes/proteins were plotted by the lowest that genes/proteins were plotted by the lowest concentration that produced a change in concentration that produced a change in gene/protein levelsgene/protein levels

Genes/proteins that produced changes at Genes/proteins that produced changes at identical concentrations were groupedidentical concentrations were grouped

Each group of genes/proteins were evaluatedEach group of genes/proteins were evaluated

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DOSE-RESPONSE FOR THE DOSE-RESPONSE FOR THE IN VITROIN VITRO EFFECTS OF ARSENIC IN IMMORTALIZED EFFECTS OF ARSENIC IN IMMORTALIZED

CELLSCELLS

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Immortalized vs. PrimaryImmortalized vs. Primary Based on the available data, immortalized cells seem Based on the available data, immortalized cells seem

to respond similarly for some endpoints; however, to respond similarly for some endpoints; however, there is limited information in the low-concentration there is limited information in the low-concentration region.region.

Some genes associated with proliferation (Some genes associated with proliferation (e.g.e.g., VEGF) , VEGF) demonstrate increased expression at lower demonstrate increased expression at lower concentrations in primary cells (1 µM versus 50 µM) concentrations in primary cells (1 µM versus 50 µM) following arsenic exposure than in immortalized cells.following arsenic exposure than in immortalized cells.

Genes associated with oxidative stress, proliferation Genes associated with oxidative stress, proliferation and apoptosis exhibited increased expression and apoptosis exhibited increased expression following acute exposure, but decreased expression following acute exposure, but decreased expression following chronic exposure in immortalized cells. following chronic exposure in immortalized cells. Similar studies were not conducted with primary Similar studies were not conducted with primary cells. cells.

Immortalized cells provide impact of duration of Immortalized cells provide impact of duration of exposure.exposure.

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Immortalized vs. PrimaryImmortalized vs. Primary

General consistency between dose-response General consistency between dose-response patterns to arsenite for primary and patterns to arsenite for primary and immortalized cells.immortalized cells.

Important because information on the impact Important because information on the impact of duration of exposure is onlyl available for of duration of exposure is onlyl available for immortalized cells (Vogt and Rossman 2001; immortalized cells (Vogt and Rossman 2001; Hu et al. 2002).Hu et al. 2002).

Increase in expression following acute Increase in expression following acute exposure (less than 72 hours); decrease exposure (less than 72 hours); decrease following chronic exposure (> 7 days).following chronic exposure (> 7 days).

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In VitroIn Vitro – Tumor-Derived – Tumor-Derived CellsCells

Majority of the information was at higher Majority of the information was at higher concentrations (> 50 µM).concentrations (> 50 µM).

Depending upon the cancer type, various cell lines Depending upon the cancer type, various cell lines may be sensitive to arsenic-induced apoptosis may be sensitive to arsenic-induced apoptosis (chemotherapy). (chemotherapy). E.g.E.g., apoptotic genes (, apoptotic genes (e.g.e.g., JNK) , JNK) may be activated at lower concentrations than in may be activated at lower concentrations than in primary cells; in the ST486 B-lymphoma cell line, primary cells; in the ST486 B-lymphoma cell line, 80% apoptosis was observed at 5µM (Muscarella and 80% apoptosis was observed at 5µM (Muscarella and Bloom 2002). Bloom 2002).

Some genes are functionally inactive in cancerous Some genes are functionally inactive in cancerous cells. In human T-cell leukemia virus type-1 (HTLV-cells. In human T-cell leukemia virus type-1 (HTLV-1), p53, critical to cell cycle control, is functionally 1), p53, critical to cell cycle control, is functionally inactive.inactive.

Some genes are over-expressed. In cervical cancer Some genes are over-expressed. In cervical cancer cells, the over-expression of Bcl-2 contributes to the cells, the over-expression of Bcl-2 contributes to the chemotherapeutic properties of arsenic.chemotherapeutic properties of arsenic.

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ConclusionsConclusions Concentration-Response information from Concentration-Response information from

primary cells provides clear evidence of primary cells provides clear evidence of concentration dependence on expressionconcentration dependence on expression Low concentrations (below 0.1 µM) – adaptive Low concentrations (below 0.1 µM) – adaptive

statestate 0.1 to 10 µM – increased expression associated 0.1 to 10 µM – increased expression associated

cell cycle control.cell cycle control. ~5 µM or greater – evidence of cytotoxicity~5 µM or greater – evidence of cytotoxicity ~1 to 10 µM – increase in genes or proteins ~1 to 10 µM – increase in genes or proteins

associated with proliferative signaling.associated with proliferative signaling. >10 µM – cell cycle stasis and apoptotic >10 µM – cell cycle stasis and apoptotic

responsesresponses

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ConclusionsConclusions

Consistency in: Consistency in:

results across cell lines (primary vs. results across cell lines (primary vs. immortalized)immortalized)

patterns across primary cell linespatterns across primary cell lines

Across cancer and noncancer responsesAcross cancer and noncancer responses

Consistency important for Consistency important for application of results in development application of results in development of a mode of actionof a mode of action

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Proposed Mode of ActionProposed Mode of Action The available The available in vitroin vitro data in primary cells data in primary cells

provide clear evidence of dose-provide clear evidence of dose-dependence of the effects of arsenic dependence of the effects of arsenic compounds on various genes or proteins compounds on various genes or proteins from concentrations of 0.005 to up to from concentrations of 0.005 to up to 1000 µM.1000 µM.

The available evidence for genomic effects The available evidence for genomic effects in the low-concentration region, combined in the low-concentration region, combined with information on the metabolism and with information on the metabolism and protein binding, supports a mode of action protein binding, supports a mode of action for inorganic arsenic carcinogenicity for inorganic arsenic carcinogenicity involving specific direct interactions with involving specific direct interactions with critical proteins overlaid against a critical proteins overlaid against a background of chemical stress.background of chemical stress.

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Proposed Mode of ActionProposed Mode of Action

Key Events appear to include:Key Events appear to include:

DNA repair inhibition under conditions DNA repair inhibition under conditions of oxdiative stress, inflammation, and of oxdiative stress, inflammation, and proliferative signaling, leading to a proliferative signaling, leading to a situation in which the cell is no longer situation in which the cell is no longer able to maintain the integrity of its DNA able to maintain the integrity of its DNA before divisionbefore division

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

INORGANIC ARSENICINORGANIC ARSENIC

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Data GapsData Gaps

Limited parallel information across Limited parallel information across species and tissue is available to conduct species and tissue is available to conduct the cross-species comparisons needed for the cross-species comparisons needed for the human risk assessment.the human risk assessment.

Additional studies are needed in the Additional studies are needed in the appropriate species following a chronic appropriate species following a chronic duration in which the tissues are brought duration in which the tissues are brought to steady state, such as subchronic to steady state, such as subchronic drinking water or inhalation studies. drinking water or inhalation studies. Acute studies provide limited information Acute studies provide limited information due to the transient nature of some gene due to the transient nature of some gene expressions.expressions.

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Ongoing ResearchOngoing Research An An in vivoin vivo study in the mouse has been study in the mouse has been

completed to evaluate changes in gene completed to evaluate changes in gene expression in the mouse bladder expression in the mouse bladder following sub-chronic exposure to following sub-chronic exposure to arsenic. arsenic.

Genomic analyses are completed in the Genomic analyses are completed in the mouse and are ongoing for human mouse and are ongoing for human bladder epithelial cells exposed bladder epithelial cells exposed in vitroin vitro at concentrations equivalent to those at concentrations equivalent to those achieved in the achieved in the in vivoin vivo study. study.

The results of these studies are The results of these studies are anticipated to be components in a anticipated to be components in a nonlinear bladder cancer risk model.nonlinear bladder cancer risk model.

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ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

ENVIRON International The HamnerAnnette Shipp Harvey ClewellDexter SullivanTracy McDonald Yager

Environmental Janice Yager

FundingEPRI