Www.apt-pharmatox.com 1 Limitations of EDSP Data Session III Assessing Cumulative Effects of...

www.apt-pharmatox.com

1

Limitations of EDSP DataLimitations of EDSP Data Session IIISession III

Assessing Cumulative Effects of Endocrine Active SubstancesAssessing Cumulative Effects of Endocrine Active Substances

The Endocrine Disruptor Screening Program:The Endocrine Disruptor Screening Program:What Can Screening Results Tell Us About Potential Adverse What Can Screening Results Tell Us About Potential Adverse

Endocrine EffectsEndocrine EffectsISRTP Workshop, Lister Hill AuditoriumISRTP Workshop, Lister Hill Auditorium

Bethesda, MD. September 9-10, 2009Bethesda, MD. September 9-10, 2009

Christopher J. Borgert, PhDChristopher J. Borgert, PhD


2

Assumptions of RPF/TEQ ApproachSafe, Environ. Health Persp. 106:1051-8.1998

• Chemical congeners;• Same molecular targets;• Same biochemical pathways;• Similar pharmacokinetic characteristics;• Identical tissue- and organ-level toxic manifestations;• Parallel dose-toxicity curves;• Non-interaction (dose additivity in mixtures);• Simplifies mixture assessment.


3

23

4

5 6

2’ 3’

5’

4’

6’

Biphenyl

Cl

Cl

Cl

Cl

Cl

3,3’,4,4’,5-PCB

Cl O

OCl Cl

Cl

2,3,7,8-TCDD

1

5

3

2O

O4 67

8

910

Dibenzo-p-dioxin

2,3,4,7,8-PeCDF

Cl

OCl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

3,3’,4,4’,5-PCBCl


4

Benzo-[a]-pyrene Dibenz-[a,h]-anthracene

Chrysene Benzo-[a]-anthracene


5o,p’-DDECl

Cl

Cl Cl

HO OH

Bisphenol-A

Cl

ClCl

OO

MethoxychlorNonylphenol

OH

TamoxifenO

N

OHH

H

H HHO

17-estradiol


6

Relative potency approaches, including dioxin TEFs, are simply dose Relative potency approaches, including dioxin TEFs, are simply dose addition applied to a specified endpoint, such as Ah receptor binding. addition applied to a specified endpoint, such as Ah receptor binding. The approach assumes that the endpoint - e.g., Ah receptor binding - is The approach assumes that the endpoint - e.g., Ah receptor binding - is a common mechanistic step in the production of all toxic effects of the a common mechanistic step in the production of all toxic effects of the chemicals to which the approach is applied.chemicals to which the approach is applied.

ConventionConvention

An estrogen equivalents (EE) approach would apply dose addition to An estrogen equivalents (EE) approach would apply dose addition to some endpoint assumed to be a surrogate for estrogen-mediated some endpoint assumed to be a surrogate for estrogen-mediated adverse effects. Direct mixture testing to confirm dose addition for adverse effects. Direct mixture testing to confirm dose addition for all potential environmental estrogens would never occur.all potential environmental estrogens would never occur.

ExtensionExtension


7

Berenbaum MC, 1981. Criteria for analyzing interactions Berenbaum MC, 1981. Criteria for analyzing interactions between biologically active agents. Advances in Cancer between biologically active agents. Advances in Cancer Research 35: 269-335Research 35: 269-335..

Now, a combination that must, by definition, always show zero Now, a combination that must, by definition, always show zero interaction interaction between agentsbetween agents is the spurious “combination” of an agent is the spurious “combination” of an agent with itself, in any arrangement of doses. This must hold, irrespective with itself, in any arrangement of doses. This must hold, irrespective of the nature of the dose-response curve of the agent or the type of of the nature of the dose-response curve of the agent or the type of effect measured. Whether the agent shows self-interaction or not, it effect measured. Whether the agent shows self-interaction or not, it is axiomatic that a combination of particular doses of one and the is axiomatic that a combination of particular doses of one and the same agent must have the same effects as the sum of those doses, same agent must have the same effects as the sum of those doses, because the “combination” and the sum are identical. (p288)because the “combination” and the sum are identical. (p288)

The TheoryThe Theory


8

What is “Expected”?2 Classical Models for Non-Interaction

Loewe Additivity[Dose Addition]

Bliss Independence [Response Addition]

• Statistical independence

• Relative effect of A not influenced by B

• Sum effects of each agent

• EA+B = EA + EB - (EA x EB)

• No self-interaction

• Agents act as simple dilutions (potency, DRC)

• Sum doses & potencies of each agent

• Da/DA + Db/DB = 1


9

Impact of the No-Interaction ModelImpact of the No-Interaction Model Borgert et al. 2004. TAAP Vol 201(2): 85-96.

Threshold Dose

D(a) D(b) D(c)

RE

SP

ON

SE

0

2

4

6

DOSE

R (a + b + c)

Dose-A

dditive

Response

-Additi

ve


10

EPA EPA CanRCanR

ILSIILSICumRCumR

EPA EPA MixMix ATSDRATSDR

TEFTEFSafe, 1998Safe, 1998

Mode of Action Classification Criteria: Mode of Action Classification Criteria: Borgert 2007. Borgert 2007. TAAP 223:TAAP 223: 114-120. 114-120.

xx xx xx xxxxMolecular targetMolecular target

xx xx xxCellular targetCellular target

xxPhysiological targetPhysiological target

xx xx xx xxxxTarget organTarget organ

xx xx xxToxic intermediatesToxic intermediates

xxCausality of stepsCausality of steps

xx xxPharmacokineticsPharmacokinetics

xx xxDetox. pathwaysDetox. pathways

xx xx xxParallel DRCsParallel DRCs

Dose AdditionDose Addition


11

Dose-Dependent Transitions in Dose-Dependent Transitions in Mechanism and InteractionMechanism and Interaction Borgert et al. 2004. TAAP Vol 201(2): 85-96.

Hydrogen cyanideHydrogen cyanide Hydrogen sulfideHydrogen sulfide

RhodenaseRhodenase

ThioThiocyanatecyanate SulfateSulfate

Sulfide oxidaseSulfide oxidase

ExcretionExcretion


12

Examples of Mechanisms Which Could Produce Examples of Mechanisms Which Could Produce Dose-Dependent TransitionsDose-Dependent Transitions

• Absorption / Distribution / Absorption / Distribution / ExcretionExcretion

• Metabolic handlingMetabolic handling

• EfficiencyEfficiency– DNA repairDNA repair– Cell killingCell killing– Rate of cell replicationRate of cell replication

• Detoxifying enzyme Detoxifying enzyme systemssystems– Modifying factorsModifying factors

Slikker et al. 2004. TAAP Vol 201(3): 203-225.

• Co-substrate depletionCo-substrate depletion

• Chemical transformation / Chemical transformation / activationactivation

• Altered homeostasisAltered homeostasis– Essential nutrientsEssential nutrients– HormonesHormones

• Repair mechanismsRepair mechanisms

• Blood flow and diffusion Blood flow and diffusion limitationlimitation


13

NSAIDs - AntithrombosisNSAIDs - Antithrombosis Renda et al., 2006. Clin. Pharmacol. & Therapeutics. 80(3):264-274.Renda et al., 2006. Clin. Pharmacol. & Therapeutics. 80(3):264-274.

Regular, Regular, but not intermittentbut not intermittent, NA-NSAID use reduces MI protection by , NA-NSAID use reduces MI protection by aspirinaspirin

• Mixed Cox 1-2 inhibitorsMixed Cox 1-2 inhibitors• Inhibition of platelet Cox 1 inhibits platelet aggregationInhibition of platelet Cox 1 inhibits platelet aggregation

Aspirin + non-aspirin-NSAIDAspirin + non-aspirin-NSAID• Increased enzyme inhibition / decreased platelet aggregation at 4 Increased enzyme inhibition / decreased platelet aggregation at 4

hours (additivity)hours (additivity)• Decreased enzyme inhibition / increased platelet aggregation at 7 Decreased enzyme inhibition / increased platelet aggregation at 7

days (antagonism)days (antagonism)• Reversible versus irreversible enzyme inhibition and threshold for Reversible versus irreversible enzyme inhibition and threshold for

TBX-ATBX-A22-mediated platelet aggregation.-mediated platelet aggregation.


Six SCs were combined at equimolar ratios and tested as a mixture at 5-6 dose levels in combination with a mixture of six PEs, also at 5-6 dose levels. Estrogenicity measured by an in vitro ER transactivation assay and an immature rat uterotrophic assay.

Determined dose of SC mixture necessary to produce an estrogenic response greater than PEs alone.

Experiment-

Charles et al. 2007. Toxicol. Appl. Pharmacol. 218: 280-288

Question- Are mixtures of estrogenic synthetic chemicals (SC) dose additive in combination with phytoestrogen (PE) mixtures?


Results- No increase in response due to the addition of SC mixture at 0.02µM and 0.2µM. Very slight increase with addition of SC mixture at 1.0µM and 2.0µM.

Clearly significant increase (p=0.006) at 3.0µM

Conclusion- SC mixture increased estrogenic response over PE background only when each chemical in the mixture was > 0.5x its individual NOEL in the estrogenic assay.

Charles et al. 2007. Toxicol. Appl. Pharmacol. 218: 280-288


16

o,p-DDTo,p-DDT p,p-DDTp,p-DDT o,p-DDEo,p-DDE p,p-DDEp,p-DDE MXCLMXCL

9.08E-03 5.68E-04 7.74E-04 0.00E-00

1.12E-041.12E-04 9.97E-059.97E-05 1.03E-041.03E-04 0.00E-000.00E-00

0.00E-00 0.00E-00 0.00E-00 0.00E-00

7.00E-067.00E-06 4.20E-064.20E-06

3.60E-05

2.00E-042.00E-04 NDND 1.00E-031.00E-03

4.00E-01 9.00E-01 8.80E-01

5.90E-065.90E-06 3.10E-063.10E-06

4.00E-01 9.00E-01

Relative Estrogenic PotencyRelative Estrogenic PotencyBorgert et al. 2003. Borgert et al. 2003. Environ. Health Persp. 111(8):Environ. Health Persp. 111(8):1020-10361020-1036


17

Relative Estrogenic PotencyRelative Estrogenic PotencyBorgert et al. 2003. Environ. Health Persp. 111(8):1020-1036

MethoxychlorMethoxychlor Bisphenol ABisphenol A1.00E-031.00E-03 1.00E-041.00E-04

3.70E-053.70E-05

NDND 1.30E-041.30E-04

3.10E-063.10E-06 1.30E-051.30E-05

8.80E-018.80E-01

8.00E-058.00E-05

4.40E-044.40E-04

1.30E-031.30E-03

9.50E-039.50E-03 2.10E-032.10E-03

1.36E-04 – 3.3E-061.36E-04 – 3.3E-06 1.20E-05 – 5.00E-061.20E-05 – 5.00E-06


Discrepancies in potency estimates are also apparent in measurements of transcription activation by phytoestrogens.

Potency estimates relative to estradiol exhibit marked discrepancies between assay systems and, in select cases, within assay systems.

When binding assays, in vitro functional assays, and in vivo end points are compared, discrepancies are evident for other phytoestrogens (e.g., coumestrol) and mycoestrogens (α-zearalenone) as well as for genistein (Whitten and Patisaul 2001).

Borgert et al. 2003. Borgert et al. 2003. Environ. Health Persp. 111(8):Environ. Health Persp. 111(8):1020-10361020-1036


19

*EEs for Natural Ligands

Estradiol 17-Estradiol 17- (E2) (E2) 1.001.00

EstriolEstriol 6.67E-026.67E-02

2-OH-E22-OH-E2 2.00E-022.00E-02

TestosteroneTestosterone 2.00E-052.00E-05

ProgesteroneProgesterone 0.000.00

Transcriptional activation of human ER - pBD-GAL4 Transcriptional activation of human ER - pBD-GAL4 reporter construct in yeast YRG2reporter construct in yeast YRG2

Chen et al., 2004. J.Biol.Chem. 279(2):33855-33864

*calculated from figure 2b


21

IFIF an Estrogen Equivalents Approach an Estrogen Equivalents Approach Were to be Undertaken, …Were to be Undertaken, …

I.I. Need rules for developing estrogen equivalents and for Need rules for developing estrogen equivalents and for applying them to the right chemicals; applying them to the right chemicals;

II.II. Need to define the hallmark estrogenic effects that confer Need to define the hallmark estrogenic effects that confer dose additive combined action dose additive combined action for adverse effectsfor adverse effects rather rather than biochemical curiosities;than biochemical curiosities;

III.III. Need to define the relevant potency ranges that fit the Need to define the relevant potency ranges that fit the dose-addition model (dose-addition model (testosteronetestosterone is NOT estrogenic) in is NOT estrogenic) in animal tests;animal tests;

IV.IV. Ideally, define a “human-relevant-potency-threshold” for Ideally, define a “human-relevant-potency-threshold” for assessing cumulative effects.assessing cumulative effects.

Www.apt-pharmatox.com 1 Limitations of EDSP Data Session III Assessing Cumulative Effects of...

Documents

Transcript of Www.apt-pharmatox.com 1 Limitations of EDSP Data Session III Assessing Cumulative Effects of...