Transcript (Q)SAR Analysis of Nongenotoxic Carcinogens

Brief Overview of the Major Mechanisms of
Nongenotoxic/Epigenetic Carcinogens and
Exploration of Possible (Q)SAR Approaches
Yin-tak Woo, Ph.D., DABT
Risk Assessment Division
Office of Pollution Prevention & Toxics
U.S. Environmental Protection Agency
Washington, DC 20460
Presented at 2nd McKim Int. QSAR Workshop
Baltimore, MD
May 8 – 10, 2012
*Disclaimer: The views expressed are solely the author’s and do not
necessarily reflect the views and policies of the U.S. EPA.
Outline
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Nongenotoxic/Epigenetic Carcinogens Defined
Importance of Identifying Nongenotoxic Carcinogens
Difficulties of Studying Nongenotoxic Carcinogens
Major Mechanisms of Nongenotoxic Carcinogens
Possible (Q)SAR Approaches
Overview of Selected Mechanisms and (Q)SAR
Integrative approaches
Conclusions and Recommendations
Defining Nongenotoxic/Epigenetic Carcinogens
• Carcinongesis is a multistage/multistep process
– Initiation: Mutation converts normal to preneoplastic cells
– Promotion: Expansion of preneoplastic cells to benign tumors
– Progression: Transformation of benign to invasive malignant tumors
• A complete carcinogen acts on all three stages
• Classification usually based on predominant mechanism
• Genotoxic carcinogens, mostly DNA-reactive, act directly on
initiation as the/a predominant mechanism
• Nongenotoxic carcinogens, act directly on promotion &/or
progression; clonally expand previously initiated cells or
trigger pathways to generate indirect genotoxic effects
Main event(s)
Key mechanistic
consideration
SAR/QSAR
mechanistic
descriptors
Initiation
Promotion
Progression
Direct DNA
binding
Indirect DNA
damage
Clonal expansion
Overcoming
suppressions (e.g.,
Electrophile,
resonance
stabilization,
nature of DNA
adduct
Receptor, cytotoxicity,
gene expression
Electrophilicity,
HOMO/LUMO,
delocalization
energies, ……
2D, 3D, docking,
biopersistence,
methylation, ….
Cell proliferation
Apoptosis
Differentiation
Homeostasis
p53, immune,
angiogenesis)
Free radical,
receptor, gene
suppression
Signal transduction, homeostasis
Reduction potential,
2D, 3D, ……
Importance of Identifying and Thoroughly
Understanding Nongenotoxic Carcinogens
• New generation products: avoid genotoxic
• Quantitative risk assessment: threshold or not,
conditional scenarios
• Human relevance/significance
• Regulatory impact
• Testing strategies for cancer bioassay: proactive,
prioritization, surrogate, weight of evidence
• Molecular biology of cancer
• Chemoprevention and treatment of cancer
Difficulties of Nongenotoxic Carcinogens
• Often target organ-, species/strain-, gender-, route-,
dose-, &/or exposure scenario-specific
• May involve multiple mechanisms/pathways
• Often indirectly genotoxic
• Some genotoxic chemicals can act via nongenotoxic
mechanisms under some scenarios or exposure
conditions
• Substantial biological understanding needed
• May require confirmatory biological studies, esp. for
regulation
(Q)SAR of nongenotoxic carcinogens
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Recently one of the most active, high incentive research areas
Multiple mechanisms with no obvious unifying concept
Often target organ-specific or cell-specific
May be > 1 molecular initiating event or pathway
(Q)SAR analysis possible for some of the mechanisms
Some mechanisms may not be of human significance
Mechanism-specific predictive biological assays may provide
supportive or confirmatory evidence
• TXG, HTS, AOP assays and Tox-21, ToxCast strategies may
provide additional input
Major nongenotoxic mechanisms
• Receptor-mediated mechanisms (AhR, PPAR, CAR, PXR, AR,
ER, other hormonal, growth factors…)
• Hormonal imbalance (thyroid, testicular, mammary, ovary,..)
• Cytotoxicity-induced cell proliferation (often organ specific)
• Oxidative Stress (ROS) and Nitrosative Stress (RNS)
• Inhibition of Intercellular Communication (GJIC)
• Perturbation of DNA methylation/gene expression
• Miscellaneous (phosphatase inhibition, choline deficiency,
immunosuppression, spindle poison, apoptosis, signal
transduction, angiogenesis, etc.)
Receptor-Mediated Mechanisms
General (Q)SAR Approaches
• Receptor-specific (e.g., nuclear vs. membrane vs
protein; well defined vs. promiscuous)
• Molecular size and shape (2D vs. 3D)
• Some may have critical regions, active site
• Mostly reversible binding
• Biological persistence of the chemical/ligand
• Measure biological half-life of the chemical/ligand
• Structural features of metabolic refractoriness
TCDD Ah Receptor Agonists SAR
- Rectangle 3 x 10 Angstroms
- Planar molecule
- Substitution at lateral positions
Cl
O
Cl
Cl
O
Cl
2,3,7,8-TCDD
Cl
Cl
Cl
O
Cl
Cl
Cl
Cl
Cl
2,3,4,7,8-PCDB
3,3',4,4',5'-PCB
NTP Tech
Report No.
Cl
Cl
Cl
Cl
Cl
Cl
Chemical Agent
Cl Cl
2,2',4,4',5,5'-HCB
NTP Bioassay Results in Female Rats
Evidence*
Target Organ(s)
Incidence**
OncoLogic
Prediction
521
2,3,7,8-TCDD
CE
Liver/lung/oral cavity
25/53
H
520
3,3’,4,4’,5’-PCB
CE
Liver/lung/oral cavity
13/53
HM
525
2,3,4,7,8-PCDB
SE
Liver/oral cavity
4/53
M
529
2,2’,4,4’,5,5’-HCB
EE
Liver
2/53
LM
*CE = clear evidence; SE = some evidence; EE = equivocal evidence
**Highest incidence observed in any one specific target organ.
Peroxisome Proliferator-Activated Receptor (PPARα) SAR
Relative peroxisome proliferative activity of chlorinated
phenoxyacetic acid in cultured hepatocytes
PPARα carcinogens: SAR and biological features
• Medium size chemicals with polar and nonpolar ends
• Polar end being carboxylic acid moiety in most cases
• Nonpolar end may be a variety of chemical structures
(e.g., branched alkyl [omega minus 1]; polyhalogenated
alkyl [esp. perfluoro]; ring-substituted phenoxy, etc.)
• Organ-, species/strain-, gender- specific
• Hepatocarcinogenicity correlates with peroxisome
proliferative activity
• Biological half-life can be an important factor due to
noncovalent binding
• Metabolic refractoriness may be important factor
CAR and PXR Ligands
Omiecinski et al. Toxicol. Sci. 120 (S1), S49-S75, 2011
Hormonal imbalance: basic principles
Hormonal imbalance: (Q)SAR approaches
• Precursor transport modifiers (e.g., ↓ iodide by
perchlorate)
• Biosynthetic pathway modifiers (e.g., ↓ thyroid
peroxidase by Amitrole, PTU; ↓ 5’-monodeiodonase
by red dye no.2)
• Catabolism of hormone by P450 inducers (e.g.,
phenobarbital, TCDD)
• Hormone secretion (e.g., ↓ thyroid by lithium?)
Some SA for thyroid carcinogens
R
R
R'
R"
N
N
H
S
N,N'-Dicyclohexylthiourea (R=H; R'=R"=C 6H11)
N,N'-Diethylthiourea (R=H; R'=R"=C 2H5)
Trimethylthiourea (R=R'=R"=CH 3)
O
HN
NH
NH-C(S)-SH
HN
NH
NH-C(S)-SH
S
S
2-Thiouracil (R=H)
6-Methylthiouracil (R=CH3)
6-n-Propylthiouracil (R=C3H7)
Ethylenethiourea
Ethylenebisdithiocarbamate
Oxidative stress of nongenotoxic carcinogens
• Structural variety: quinones and quinoids, aromatic amines,
polyhalogenated hydrocarbons, oxidants, transition metal
compounds, peroxy compounds, etc.
• Free radicals, reactive oxygen species, lipid peroxides,
malondialdehyde, etc. as secondary reactants
• Free radical stabilization may be a factor for some (SAR)
• 8-Hydroxy/8-Oxo-2’-deoxyguanosine an important biomarker
for indirect DNA damage
• Lipid peroxides and malondialdehydes measurable by
thiobarbituric acid (TBA) assay
• Antioxidants and free radical scavengers protective
Formation of 8-oxo-dG and 8-OHdG in oxidative stress
(from Valavanidis et al. J. Env.Sci. Hlth. C27, 120, 2009)
Intercellular Communication (GJIC) Inhibition
as a Nongenotoxic carcinogenic mechanism
CF3(CF2)nCOOH
CF3(CF2)nSO3H
n= 0 to 3,14,or 16, inactive; n= 4, weak; n=5-8, active
n=3, inactive; n=5 or 7, active
Perturbation of DNA methylation/gene expression
• Methylation of cytosine at 5-position regulates gene
expression
• Altered DNA methylation may lead to carcinogenesis
• Global vs. regional
• Alteration of methyl donor (SAM) pool (e.g., ↓ As, 5azadeoxycytidine; ↑ methionine, choline, etc.)
• Manipulation of methyltransferase
• Microarray-based transcriptional studies
Cytotoxicity-induced regenerative cell proliferation
• Male rat kidney tumors via α2µnephropathy (e.g., tert-butyl
alcohol, jet fuels, unleaded gasoline)
• Bladder stones, calculi, microcrystals (e.g., NTA, saccharin,
melamine, sulfonamides, Fosetyl)
• Liver tumors via “chemical hepatectomy” (e.g., carbon
tetrachloride, chloroform)
• Spleen tumors via splenotoxicity/hemosiderosis (e.g.,
hemolytic aromatic amines and azodyes)
• Nasal tumors via in situ formation of reactive chemicals or
acids (e.g., Alachlor, vinyl acetate)
• Persistent portal of entry (e.g., lung, skin) exposure to acids or
irritants (e.g., sulfuric acid mist, petrol distillates)
Cytotoxicity via Alpha-2µ Nephropathy
Criteria for classification
• The renal tumors occur only in male rats
• Acute exposure cause hyaline droplet in proximal
convoluted tubules (P2-segment)
• Protein in hyaline droplet = α2µ-globulin
• Observation of Hallmark histopath lesions (granular
casts, linear papillary mineralization)
• Absence of hyaline droplets and histopath in female
rats and mice of both sexes
• Negative in short term tests for genotoxicity
Searching for a common mechanistic basis
Small tert-alcohol as potential SA for
male rat kidney α2µnephropathy
CH3
CH3
CH3-CH-CH2-C-CH3
CH3
O
CH3-CH-CH2-C-CH3
CH3
Isooctane (TMP)
CH3
CH3
CH3-C-CH2-C-CH3
OH
CH3
2-OH-TMP
CH3
MTBE
O
CH3-C-CH2-C-CH3
OH
major metabolite
of MIBK
CH3
CH3-CH-O-C-CH3
CH3-C-CH3
O-CH3
MIBK
CH3
HOH2C
CH3
PG t-Butyl Ether
CH3
CH3-C-CH3
OH
t-Butyl alcohol
Cytotoxicity induced nasal tumors
Cytotoxicity Induced: hepatotoxicity via Inhibition of
Protein Phosphates 1, 2A (from Dr. Fujiki’s lab)
Microcystin-LR: chemical structure and SAR
(red: Adda region; green : 6(Z)- stereoisomer)
Binding of Okadaic Acid and Microcystin-LR
to Protein Phosphate 2A Core Enzyme
(from Xing et al., Cell 127, 341, 2006)
Integrative Approach
Initiation
(e.g., Electrophilicity, DNA
adduct, genotox, transgenic
rodent models, ras, etc.)
Promotion
(e.g, cell proliferation,
apoptosis, gap junct.
cyp, hormonal imbal,
gene expression,
mitogenesis,
ppar, myc, etc.)
Woo et al. (1998)
Progression
(e.g, immune
suppression, free
radical, metastasis,
angiogenesis, etc.)
EPA gene expression and pathway analysis study of
nongenotoxic carcinogenic conazole pesticides
(from Hester et al. Tox. Sci. 127, 54, 2012)
F
Cl
OH
Cl
Cl
Cl
O
O
O
N
N
Cyproconazole
N
N
N
N
N
N
Epoxiconazole
N
Propiconazole
Highlights from the study
• Microarray-based transcriptional analysis showed a
common basis
• 330 common altered genes for Cyp, GSH Stransferase and oxidative stress
• Subset of 80 altered genes associated with cancer.
• Pathways associated: xenobiotic metabolism,
oxidative stress, cell signaling, and cell proliferation.
• Common TGFa-centric pathway provides a more
refined toxicity profile
TGFα-mediated cell proliferation as the common
mechanistic pathway for Cypro, Epoxi and Propi
Cancer Hallmark processes* and pathways**
• Inducing Angiogenesis (VEGF, IGF-1R, miRNA)
• Resisting cell death (apoptosis, p53, DNA damage)
• Sustaining proliferative signaling (growth factors, Akt, MAP
kinase, oncogenes)
• Evading growth suppressors (p53, apoptosis, TGFβ, EMT)
• Enabling cell immortality (telomerase, p53,.DNA repair)
• Activating invasion and metastasis (EMT, tumor metastasis)
• Evading Immune destruction (T- & B-cell, inflammatory
response)
• Reprogramming energy metabolism (hypoxia, glycosylation)
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*Hanahan and Weinberg, Cell, 144,646 (2011). **see “Pathway Central” online.
EPA ToxCast study mapping assays to cancer hallway processes ongoing
Suggestions for (Q)SAR of
Nongenotoxic carcinogens
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Expansion of database (e.g., OPP, pharma)
Structural and Functional Classifications
Structural alerts and modification factors
Integrating with HTS, TXG, pathway analysis
Defining conditions and scenarios
Building consensus and testing strategies
Beware of real life exposure and susceptible
subpopulations