Genetic Toxicity Mini-Adverse Outcome Pathways (Mini-Aops)
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Genetic Toxicology Mini-Adverse Outcome Pathways (mini-AOPs) Azeddine Elhajouji March 22, 2018 Outline ❖ Introduction to AOP ❖ Mode of action and the need for mini-AOPs concept ❖ Mini-AOPs in genetic Toxicology ❖ Examples of mini-AOPs 2 ADVERSE OUTCOME PATHWAYS (AOP) • An Adverse Outcome Pathway (AOP) is a structured representation of biological events leading to adverse effects that is considered relevant to risk assessment. • The AOP links in a linear way existing knowledge along a pathway of causally connected key events (KE) between 2 points – a molecular initiating event (MIE) and an adverse outcome (AO). • The events occur at levels of biological organisation relevant to risk assessment e.g. molecular, cellular, tissue/organ, organism and/or population. • The linkage between the events is described by key event relationships (KERs) that describe the scientifically established causal relationship between the chain of key events. 3 ADVERSE OUTCOME PATHWAYS Molecular Cellular Organ Organism Population Ref: Ankley et al. (2010) Environ. Toxicol. Chem. 29: 730-741 KER1 KER2 KER3 KER4 MIE KE1 KE2 KE3 AO 4 “THE NEED FOR mini-AOPs CONCEPT” BETTER UNDERSTANDING OF MOA positive in standard gene mutagen what is the MOA mammalian clastogen leading to the GeneTox assay aneugen genotoxic outcome? • genetox assays good for hazard identification • not designed to provide detailed mechanistic information • do not identify molecular target • have often unclear structure activity relationship information • are not designed to provide dose-response information 5 WHY MINI-AOP AND NOT AOP? Exposure Exposure Molecular Organelle/ Cellular Tissue Organ Individual Population Source Initiating Molecular Effects Effects Effects Effects Effects Event Effects Toxicity Pathway (mini-AOP) Adverse Outcome Pathway • In vitro cellular effects can be more easily measured – More information available – Easier to hit dose-response relationships – Easier to address data gaps • Tissue effects often a combination of different cellular effects • Adverse health outcomes (i.e. cancer) do not always have a clear relationship – not clear that tubulin inhibitors initiate cancer – most genotoxins require significant toxicity before you see genotoxicity – threshold effects – sensitivity of cancer bioassay? 6 mini-AOPs DEFINITIONS A specialized type of key event that represents the initial point of chemical Molecular MIE interaction on the molecular level within the cell that results in an event that initiating event starts the mini-AOP. A change in biological state that is both measurable and essential to the Key event progression of a defined biological perturbation leading to a specific KCE genotoxic outcome. A scientifically-based relationship that connects one key event to another, defines a directed relationship between the two (i.e., identifies one as Key event KER upstream and the other as downstream), and facilitates inference or relationship extrapolation of the state of the downstream key event from the known, measured, or predicted state of the upstream key event. A specialized type of key event that is generally accepted as being of Genotoxic GO significance for the genomic integrity of the cell and is expected to result Outcome an apical endpoint in an accepted regulatory guideline toxicity test. Indirect KER Indirect KER KER1 KER2 KER3 KER4 MIE KCE1 KCE2 KCE3 GAO Indirect KER 7 https://aopwiki.org/aops?utf8=%E2%9C%93&search=tubulin&commit=Search WEIGHT OF EVIDENCE FOR KEY EVENT RELATIONSHIPS Evolved Bradford Hill Defining Questions Considerations Biological Concordance Does the hypothesized AOP conflict with broader biological knowledge? How well established is the AOP? Essentiality of Key Events Is the sequence of events reversible if dosing is stopped or a key event prevented? Concordance of Empirical Dose response ‒ Are the key events observed at doses Observations below or similar to those associated with the apical effect? Temporality ‒ Are the key events observed in hypothesized order? Incidence ‒ Is the frequency of occurrence of the adverse effect less than that for the key events? Consistency Is the pattern of effects across species/strains/organs/test systems what would be expected based on the hypothesized AOP? Analogy Would the mode of action be anticipated based on broader chemical specific knowledge? 8 Meek et al., 2014 MOLECULAR INITIATING EVENTS IN GENOTOXICITY Aneugenic MOA Clastogenic MOA • Tubulin binding • DNA alkylation • Aurora inhibition • Topoisomerase I & II • Polo kinase inhibition inhibition • CDK 1 inhibition • DNA intercalation • NEK2 inhibition • Nucleoside analogues • Actin depolymerization • Reactive oxygen species • HSP90 inhibition • PARP Inhibition • HDAC inhibition • DNA Repair inhibition • Oxidative DNA damage 9 Example of mini-AOPs: Tubulin Binders & Topoisomerase II Inhibitors 10 MITOSIS OVERVIEW 11 http://fhs-bio-wiki.pbworks.com/w/page/12145788/Mitosis MICROTUBULI & TUBULIN BINDING SITES Colchicine [approved for acute goat] NPI-2358 [Phase III] CA-4PABT-751 [Phase II] α-Tubulin β-Tubulin Tubulin Dimer Oxi4503, SCB01A, CKD-516 [Phase I] 8 nm Paclitaxel, Docetaxel, Cabazitaxel Laulimalide [preclinical] [approved] Peloruside A [preclinical] TPI-287 [Phase II] Opaxio [Phase II] Ortataxel [Phase II] Vinblastine, Vincristine, Eribulin [approved] Ixabepilone [approved] Vindesine [Phase III] ALB-109564 [Phase I] Tubulin Subunit - (minus) end + (plus) end Stabilizing Binders Destabilizing Binders Taxanes Colchicine Epothilones Vincaalkaloids Laulimalide MICROTUBULE Kunal Nepali et al. Expert Opinion on Investigational 12 Drugs 2016;25(8):917-936 TUBULIN BINDER ADVERSE OUTCOME PATHWAY Marchetti et al., Environ Mol Mutagen 2016 ;57(2):87-113 13 mini-AOP: TUBULIN BINDERS & ANEUPLOIDY INDUCTION MIE KCE1 KCE2 KCE3 KCE4 GAO Tubulin Binding to depolymerization tubulin /stabilization Mitotic arrest Abnormal spindle formation Incorrect Non- chromosome Disjunction alignment & & MN 14 segregation mini-AOP : TUBULIN BINDERS & ANEUPLOIDY INDUCTION ASSAYS AND TOOLS (RED BOX) MIE KCE1 KCE2 KCE3 KCE4 GAO Tubulin Binding to Tubulin depolymerization polymerization tubulin /stabilization assay Mitotic Quantify H3 arrest positive cells Abnormal FISH with pan- spindle centromeric formation probe Incorrect Non- Immuno- chromosome Disjunction histochemistry alignment & & MN 15 segregation mini-AOP : TUBULIN BINDERS INDUCE MULTIPLE GAOs MIE KCE1 KCE2 KCE3 KCE4 GAO Tubulin Abnormal Binding to Mitotic depolymerization 1 spindle tubulin arrest /stabilization formation 2 BCL2/MCL1 3 degradation Incorrect chromosome alignment & Cyclin B1 D 2b Caspase segregation degradation activation 2a Non- Polyploidy Clastogenic Mitotic Disjunction induction damage Death & MN 16 TUBULIN BINDERS KEY EVENT RELATIONSHIPS –KERS- Level of KCE (upstream) Relationship type KCE (downstream) Confidence in KER Microtubuli de- Tubulin Binding Directly leads to strong polymerization/stabilization Microtubuli de- Directly leads to Extended Mitotic arrest strong polymerization/stabilization Extended mitotic arrest Leads to Mitotic abnormalities medium Chromosomal loss and non- Mitotic abnormalities Directly leads to strong disjunction Extended mitotic arrest Leads to Caspase activation strong Mitotic death and/or Caspase activation Leads to medium clastogenicity Extended mitotic arrest Leads to Cyclin B1 degradation strong Cyclin B1 degradation Directly Mitotic slippage strong 17 Mini-AOP: Topoisomerase II Inhibition Leads to Clastogenicty & Tranlocations 18 Functions of topoisomerases II ▪ Resolve the problems associated with the topological constraints of the genetic material (i.e., DNA under- or overwinding, knotting, and tangling) ▪ Transiently cleave both strands of the double helix ▪ Play essential roles in a number of genetic processes, including DNA replication, transcription, and recombination, as well as chromosome segregation. 19 Two main mechanisms of topoisomerases II inhibitors ▪ Topoisomerase II poisons kill cells – By increasing levels of covalent topoisomerase II-cleaved DNA complexes – By interfering with the ability of the enzyme to religate cleaved DNA molecules → Resulting in [stabilization of] cleaved DNA ▪ Topoisomerase II catalytic inhibitors – By robbing the cell of the essential catalytic functions of the type II enzymes – By a variety of steps of the topoisomerase II catalytic cycle, including [inhibition of] DNA cleavage → Resulting in inhibition of Topoisomerase II functions and inhibition of DNA cleavage. 20 TOPOISOMERASE POISONS Binding to DNA-topoisomerase II complex leading to genotoxicity (clastogenic lesions, gene mutations) MIE KCE1 KCE2 KCE3 GAO Inhibition of Increase in the the ability to number and religate after lifetime of Clastogenic cleavage cleavage lesions Binding to (cleaved DNA) complexes DNA Double DNA-topo II Strand Breaks Stabilization of complex Blockade of the Translocation/ the cleavage- replication forks religation Mutations and transcription complex machinery (cleaved DNA) 21 Topoisomerase Poisons Stressors MIE KCE1 KCE2 KCE3 GAO Drugs: Etoposide Amsacrine Ametantrone Zagotto et al 2014 Inhibition of Increase in the the ability to number and religate after lifetime of Clastogenic cleavage cleavage lesions Binding to (cleaved DNA) complexes DNA Double DNA-topo II Strand Breaks Stabilization of complex Blockade of the Translocation/ the cleavage- replication forks religation Mutations and transcription complex machinery (cleaved DNA) Drugs: Xanthocidin Takeda et al 22 2014 TOPOISOMERASE POISONS ASSAYS/TOOLS MIE KCE1 KCE2