AOP ID and Title:

Short Title: MEK-ERK1/2 activation leading to deficits in learning and cognition via ROS

Authors

Of the originating work:

Katherine von Stackelberg (Harvard Center for Risk Analysis, Boston, MA, USA.) (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Elizabeth Guzy (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Tian Chu (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)

Birgit Claus Henn (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.) (Department of Environmental Health, Boston University School of Public Health, Boston, MA, USA.)

Of the content populated in the AOP-Wiki:

Travis Karschnik (General Dynamics Information Technology, Duluth, MN, USA.)

Status

Abstract

Metal mixture activation of ERK1/2 and JNK1/2 in astrocytes leads to increased Ca2+ release (Asit Rai et al., 2010).  Alterations to calcium, an essential nutrient which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, and neurotransmission can be expected to have downstream effects in those functions (Antonio et al., 2002).  While a variety of stimuli can trigger opening of the mitochondrial transition pore and cause apoptosis, a sustained intracellular increase in Ca2+ is one of the better-known triggers (Mattson 2000).  Mitochondria play a role in stress responses and can produce ROS when damaged. Mitochondria are indeed a major source of ROS (Yan et al., 2013).  Unchecked, excessive ROS can lead to the destruction of cellular components including lipids, protein, and DNA, and ultimately cell death via apoptosis or necrosis (Kannan and Jain 2000).  Aberrant apoptosis has been implicated in the  pathogenesis of neurodegeneration (Okouchi et al., 2007).  It is well accepted that impairment of cell function or cell loss (neurodegeneration) in hippocampus will interfere with memory processes, since the hippocampus plays a key role in memory (Barker and Warburton 2011).

MEK-ERK1/2 is important in understanding uptake of metals into the brain and its relationship to deficits in learning and cognition from exposure to metals commonly detected at Superfund sites including lead, cadmium, manganese, and arsenic.  Current risk assessment guidance dictates a largely chemical-by-chemical evaluation of exposures and risks, which fails to adequately address potential interactions with other chemicals, nonchemical stressors, and genetic factors. Cumulative risk assessment methods and approaches are evolving to meet regulatory needs (MacDonell et al., 2013; Backhaus and Faust 2012; IPCS 2009), but significant challenges remain. As our understanding of complex exposures and interactions continues to grow, synthesis and integration across disciplines and studies focused on different aspects of the environmental fate–exposure–toxicology–health outcome continuum are required to assess the likelihood of adverse effects and to support cumulative risk assessment.  Environmental exposures are virtually always to complex mixtures (von Stackelberg et al., 2015).

 

Background

An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications.  In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood (Schubert et al., 2013; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption.  This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

1. Support for Biological Plausibility of KERs	Defining Question	High (Strong)	Moderate	Low (Weak)
	Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?	Extensive understanding of the KER based on extensive previous documentation and broad acceptance.	KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete	Empirical support for association between KEs , but the structural or  functional relationship between them is not understood.
Relationship 2942: Activation of MEK, ERK1/2 (2146) leads to Increase, intracellular calcium (1339)	Moderate Empirical evidence indicates a complex relationship between MEK, ERK1/2 activation and inhibition and Ca2+ response including Ca2+ feeding back into a ERK1/2 activation.  This relationship appears to vary across species and cell type.
Relationship 3140: Increase, intracellular calcium (1339) leads to N/A, Mitochondrial dysfunction 1 (177)	Moderate There are both accepted associations between these two KEs and empirical evidence but the current state of understanding falls short of extensive.
Relationship 3141: N/A, Mitchondrial dysfunction 1 (177) leads to Increased, Reactive oxygen species (1115)	High This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.
Relationship 2966: Increased, Reactive oxygen species (1115) leads to Apoptosis (1262)	High This is a well-studied relationship across taxa where modulation of ROS and its effect on subsequent apoptosis has been examined.
Relationship 2967: Apoptosis (1262) leads to N/A, Neurodegeneration (352)	High This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.
Relationship 1069: N/A, Neurodegeneration (352) leads to Impairment, Learning and memory (341)	High This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.
Relationship 2968: Increase, intracellular calcium (1339) leads to Apoptosis (1262)

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life Stage

Life stages applicable to this AOP encompass the full life cycle.  Many of the key events are measured in pregnant females with the adverse outcome (impairment, learning and memory) measured at all life stages.

Taxonomic Applicability

Most evidence for this AOP is derived from rodents and humans where rodents were selected with their ability to model human responses.

Sex Applicability

This AOP is applicable to all sexes.

Essentiality of the Key Events

2. Essentiality of KEs	Defining question	High (Strong)	Moderate	Low (Weak)
	Are downstream KEs and/or the AO prevented if an upstream KE is blocked?	Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs	Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE	No or contradictory experimental evidence of the essentiality of any of the KEs.
KE 2146: Activation of MEK, ERK1/2	Moderate MEK, ERK1/2 activation is fundamental in delivering signals which regulate the cell cycle, proliferation, differentiation, adhesion, and more.  Disruptions in this activation have wide reaching effects however, there is evidence that downstream KEs can also activate this KE.
KE 1339: Increase, intracellular calcium	High Calcium, as a primary intracellular messenger in neurons and regulator of cell responses to stress has been shown to play an integral role in subsequent KEs.
KE 177: N/A, Mitchondrial dysfunction 1	High The ubiquity and role of mitochondria in cell function is such that changes in this KE necessitate changes in downstream KEs.
KE 1115: Increased, Reactive oxygen species	High ROS has been shown to mediate apoptosis across taxa with changes in ROS levels affecting subsequent apoptosis.
KE 1262: Apoptosis	High Unregulated apoptosis has been shown to affect neurodegeneration and eventual learning and memory tasks in human and rodent models.
KE 352: N/A, Neurodegeneration	High Neurodegeneration has been causally linked to learning and memory tasks in human and rodent models.
KE 341: Impairment, Learning and memory	N/A
AOP 500	High/Moderate There is evidence for manipulation of downstream KEs based on manipulation of upstream KEs in multiple KERs.

Weight of Evidence Summary

3. Empirical Support for KERs	Defining Questions	High (Strong)	Moderate	Low (Weak)
	Does empirical evidence support that a  change in KEup leads to an appropriate change in KEdown? Does KEup occur at  lower doses and earlier time points than KE  down and is the incidence of KEup > than  that for KEdown? Inconsistencies?	if there is dependent change in both events  following exposure to a wide range of specific stressors (extensive evidence for temporal, dose- response and incidence concordance) and no or  few data gaps or conflicting data	if there is demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with the expected pattern that can  be explained by factors such as experimental design, technical considerations, differences  among laboratories, etc.	if there are limited or no studies reporting dependent change in both events following  exposure to a specific stressor (i.e., endpoints never measured in the same study  or not at all), and/or lacking evidence  of temporal or dose- response concordance, or identification of significant inconsistencies in empirical  support across taxa and species that don’t align with the expected pattern for the hypothesised AOP
Relationship 2942: Activation of MEK, ERK1/2 (2146) leads to Increase, intracellular calcium (1339)	Moderate The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.  Heavy metals like cadmium can complicate issues related to calcium levels since the metal itself can act in place of calcium in cell function.
Relationship 3140: Increase, intracellular calcium (1339) leads to N/A, Mitchondrial dysfunction 1 (177)	Moderate The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures.  Some inconsistency was documented in the relationship between the two events regarding which preceded the other in different taxa and cell types. Heavy metals like cadmium can complicate issues related to calcium levels since the metal itself can act in place of calcium in cell function.
Relationship 3141: N/A, Mitchondrial dysfunction 1 (177) leads to Increased, Reactive oxygen species (1115)	High The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.
Relationship 2966: Increased, Reactive oxygen species (1115) leads to Apoptosis (1262)	High The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.
Relationship 2967: Apoptosis (1262) leads to N/A, Neurodegeneration (352)	High The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.  There are also numerous studies investigating this relationship in the context of neurodegenerative diseases.
Relationship 1069: N/A, Neurodegeneration (352) leads to Impairment, Learning and memory (341)	High The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. There are also numerous studies investigating this relationship in the context of neurodegenerative diseases.
Relationship 2968: Increase, intracellular calcium (1339) leads to Apoptosis (1262)	High The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.

Considerations for Potential Applications of the AOP (optional)

Developmental neurotoxicity (DNT) is an adverse outcome of concern to multiple regulatory agencies. In vitro screening assays for MEK-ERK1/2 activation would not be recommended as a direct alternative or replacement to established DNT assays like OECD Test No. 426 (OECD 2007). However, detection of MEK-ERK1/2 activation in neuronal cell types may be used to prioritize chemicals with potential to elicit neurotoxicity and flag them for testing in ortogonal assays for evaluating DNT, including proposed alternative test methods (Bal-Price et al. 2018; Crofton et al 2022).  

References

Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266

Backhaus T, Faust M. Predictive environmental risk assessment of chemical mixtures: A conceptual framework. Environmental Science & Technology, 2012; 46(5):2564–2573.

Bal-Price A, Crofton KM, Sachana M, Shafer TJ, Behl M, Forsby A, Hargreaves A, Landesmann B, Lein PJ, Louisse J, Monnet-Tschudi F, Paini A, Rolaki A, Schrattenholz A, Sunol C, van Thriel C, Whelan M, Fritsche E. Putative adverse outcome pathways relevant to neurotoxicity. Critical Reviews in Toxicology, 2015; 45(1):83–91.

Bal-Price A, Hogberg HT, Crofton KM, Daneshian M, FitzGerald RE, Fritsche E, Heinonen T, Hougaard Bennekou S, Klima S, Piersma AH, Sachana M, Shafer TJ, Terron A, Monnet-Tschudi F, Viviani B, Waldmann T, Westerink RHS, Wilks MF, Witters H, Zurich MG, Leist M. Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity. ALTEX. 2018;35(3):306-352. doi: 10.14573/altex.1712081. Erratum in: ALTEX. 2019;36(3):506. 

Barker GR, Warburton EC. 2011. When is the hippocampus involved in recognition memory? J Neurosci 31(29): 10721-10731.

Crofton KM, Bassan A, Behl M, Chushak YG, Fritsche E, Gearhart JM, Marty MS, Mumtaz M, Pavan M, Ruiz P, Sachana M, Selvam R, Shafer TJ, Stavitskaya L, Szabo DT, Szabo ST, Tice RR, Wilson D, Woolley D, Myatt GJ. Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches. Comput Toxicol. 2022 May;22:100223. doi: 10.1016/j.comtox.2022.100223. 

International Programme on Chemical Safety (IPCS),World Health Organization (WHO). Assessment of combined exposures to multiple chemicals. Report of a WHO/IPCS  International Workshop, 2009.

Kannan, K, Jain, SK. Oxidative stress and apoptosis. Pathophysiology. 2000. 7:153-163.

Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. "Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework," Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.

M.Teresa Antonio, Noelia López, M.Luisa Leret, Pb and Cd poisoning during development alters cerebellar and striatal function in rats, Toxicology, Volume 176, Issues 1–2, 2002,
Pages 59-66, ISSN 0300-483X, https://doi.org/10.1016/S0300-483X(02)00137-3

MacDonell MM, Haroun LA, Teuschler LK, Rice GE, Hertzberg RC, Butler JP, Chang Y-S, Clark SL, John AP, Perry CS, Garcia SS, Jacob JH, Scofield MA. 2013. Cumulative risk assessment toolbox:Methods and approaches for the practitioner. Journal of Toxicology, 2013; Article ID 310904, doi:10.1155/2013/310904.

Mattson, M. Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1, 120–130 (2000). https://doi.org/10.1038/35040009

OECD (2007), Test No. 426: Developmental Neurotoxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264067394-en.

Okouchi, Masahiro, et al. "Neuronal apoptosis in neurodegeneration." Antioxidants & redox signaling 9.8 (2007): 1059-1096.

Schubert D, Martens GJM, Kolk SM. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders. Molecular Psychiatry, 2013; 2014:1–15.

Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.

Appendix 1

List of MIEs in this AOP

Event: 2146: Activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase 1/2

Short Name: Activation of MEK, ERK1/2

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

ERK1 and ERK2 are proteins of 43 and 41 kDa that are nearly 85% identical overall, with much greater identity in the core regions involved in binding substrates (Boulton et al., 1990; 1991). The two phosphoacceptor sites, tyrosine and threonine, which are phosphorylated to activate the kinases, are separated by a glutamate residue in both ERK1 and ERK2 to give the motif TEY in the activation loop (Payne et al., 1991). Both are ubiquitously expressed, although their relative abundance in tissues is variable. For example, in many immune cells ERK2 is the predominant species, while in several cells of neuroendocrine origin they may be equally expressed (Gray Pearson and others 2001). They are stimulated to some extent by a vast number of ligands and cellular perturbations, with some cell type specificity (Lewis et al., 1998). In fibroblasts (the cell type in which the generalizations about their behavior and functions have been developed) they are activated by serum, growth factors, cytokines, certain stresses, ligands for G protein-coupled receptors (GPCRs), and transforming agents, to name a few (Gray Pearson and others 2001). They are highly expressed in postmitotic neurons and other highly differentiated cells (Boulton et al., 1991). In these cells they are often involved in adaptive responses such as long-term potentiation (English and Sweatt 1996; Atkins et al., 1998; Rossi-Arnaud et al., 1997).

How it is Measured or Detected

Western blotting and immunoblotting.

References

Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD 1998 The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1 :602 –609

Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65 :663 –675

Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH 1990 An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249 :64 –67

English JD , Sweatt JD 1996 Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271 :24329 –24332

Gray Pearson and others, Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions, Endocrine Reviews, Volume 22, Issue 2, 1 April 2001, Pages 153–183, https://doi.org/10.1210/edrv.22.2.0428

Lewis TS, Shapiro PS, Ahn NG 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74 :49 –139

Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shananowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10 :885 –892

Rossi-Arnaud C, Grant SG, Chapman PF, Lipp HP, Sturani E, Klein R 1997 A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature 390 :281 –286

List of Key Events in the AOP

Event: 1339: Increase, intracellular calcium

Short Name: Increase, intracellular calcium

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

Calcium is arguably the most versatile and important intracellular messenger in neurons (Berridge et al., 2000). Interestingly, although calcium may often promote neuronal death, it can also activate pathways that promote survival. For example, calcium can promote survival through a pathway involving activation of protein kinase B (PKB/Akt) by calcium/calmodulin-dependent protein kinase (Yano et al., 1998). Calcium is a prominent regulator of cellular responses to stress, activating transcription through the cyclic-AMP response element-binding protein (CREB), which can promote neuron survival in experimental models of developmental cell death (Hu et al., 1999). Calcium can also activate a rapid neuroprotective signalling pathway in which the calcium-activated actin-severing protein gelsolin induces actin depolymerization, resulting in suppression of calcium influx through membrane NMDA (N-methyl-d-aspartate) receptors and voltage-dependent calcium channels (Furukawa et al., 1997). This may occur through intermediary actin-binding proteins that interact with NMDA receptor and calcium channel proteins. Finally, signals such as calcium and secreted amyloid precursor protein-α (sAPP-α), which increase cyclic GMP production, can induce activation of potassium channels and the transcription factor NF-κB, and thereby increase resistance of neurons to excitotoxic apoptosis (Furukawa et al., 1996).

How it is Measured or Detected

An increase in [Ca^2+]i was measured using Fluo3 AM as an indicator dye after the addition of metals (single or in mixture) to the culture wells following an optimized protocol (Arey et al., 2022). The fluorescent signals were read by fluorescence imaging plate reader Synergy HT (BioTek, Winooski, VT) (Rai and others 2010).

Briefly, Ca2+ levels in human astrocytes were monitored by fluorescence microscopy using the Ca2+ indicator fluo-4. Slices were incubated with fluo-4-AM (2–5 µL of 2 mM dye were dropped over the tissue, attaining a final concentration of 2–10 µM and 0.01% of pluronic) and Sulforhodamine 101 (100 µM) for 30–60 min at room temperature (Navarrete and others 2013). In these conditions, most of the Fluo-4-loaded cells were astrocytes as indicated by their SR101 staining (Nimmerjahn et al., 2004; Dombeck et al., 2007; Kafitz et al., 2008; Takata and Hirase 2008), and confirmed in some cases by their electrophysiological properties. Astrocytes were imaged with an Olympus FV300 laser-scanning confocal microscope or a CCD camera (Retiga EX) attached to the Olympus BX50WI microscope (Navarrete and others 2013).

Diversity of endogenous Ca2+ activity in a mature hippocampal astrocyte in situ: Ca2+ signals in cell body and processes are different. (A) Cumulative Ca2+ activity recorded in an astrocyte over a 165 s period revealed by the calcium indicator Fluo4-AM. The visible boundaries of the astrocyte are shown in white. Note the different intensities of spatially-
confined local activity in the astrocyte cell body (s), primary process (p1) stemming from the soma and secondary processes (p2) branching from a primary process. Intensity of the
normalized cumulative activity is expressed in arbitrary units (a.u.) and shown in pseudocolour, from dark (lowest) to white (highest). (B) Frequency map of the Ca2+ activity in the astrocyte during the 165 s period as in A. Activity is measured in individual pixels, expressed in mHz and color-coded from black (never active) to dark red (frequently active). Most of the activity is within the white boundaries and the most frequently active pixels are in defined small regions (arrowheads) of the primary and secondary processes (30 mHz), whereas pixels of the soma are less active (~10 mHz) (Volterra et al., 2014). 

Free intracellular calcium ions were measured using the fluorescent calcium indicator FLUO-3/AM (Molecular probes, Eugene, OR, USA). Cells (4 × 10⁴ cells/cm²) were seeded in 24-well plates for 24 h to reach 60%–70%, and then treated for 24 h with As(III) (0.5 and 1 mg/l), or coexposed to As(III) (1 mg/l) and F (2.5, 5, and 10 mg/l). After treatment, supernatant was collected and combined with trypsinized cells. Pelleted samples were resuspended in 500 μl of FLUO-3/AM (4 μmol/l) and incubated at 37 °C for 30 min. After centrifugation, cells were washed with HBSS (Hank's Buffered Salt Solution, Sigma), made up to 400 μl with HBSS and analyzed by flow cytometry. The signal from FLUO-3/AM bound to Ca²⁺ was recorded using the Fl-1 channel (Rocha et al., 2011).

Fluo-4/AM was used as an intracellular free Ca²⁺ fluorescent probe to analyze [Ca²⁺]_i in Cd-exposed cerebral cortical neurons. In short, the harvested cells were incubated with Fluo-4/AM (5 µmol/L final concentration) for 30 min at 37°C in the dark, washed with PBS, and analyzed on a BD-FACS Aria flow cytometry. Intracellular [Ca²⁺]_i levels were represented by fluorescent intensity. Fluorescent intensity was recorded by excitation at 494 nm and emission at 516 nm. The data were analyzed by Cell Quest program (Becton Dickinson), and the mean fluorescence intensity was obtained by histogram statistics (Yuan et al., 2013).

References

Arey BJ Seethala R Ma Z Fura A Morin J Swartz J Vyas V Yang W Dickson JK JrFeyen JH A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo Endocrinology 2005 146 2015 2022

Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266

Berridge, M. J., Lipp, P. & Bootman, M. D. The versatility and universality of calcium signaling. Nature Rev. Mol. Cell Biol. 1, 11– 21 (2000).

Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW. Imaging large-scale neural activity with cellular resolution in awake, mobile mice, Neuron, 2007, vol. 56 (pg. 43-57)

Furukawa, K. et al. The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178– 8186 (1997).

Furukawa, K., Barger, S. W., Blalock, E. M. & Mattson, M. P. Activation of K+ channels and suppression of neuronal activity by secreted β-amyloid-precursor protein. Nature 379, 74–78 (1996).

Hu, S. C., Chrivia, J. & Ghosh, A. Regulation of CBP-mediated transcription by neuronal calcium signaling. Neuron 22, 799– 808 (1999).

Kafitz KW, Meier SD, Stephan J, Rose CR. Developmental profile and properties of sulforhodamine 101-labeled glial cells in acute brain slices of rat hippocampus, J Neurosci Methods, 2008, vol. 169 (pg. 84-92)

Marta Navarrete and others, Astrocyte Calcium Signal and Gliotransmission in Human Brain Tissue, Cerebral Cortex, Volume 23, Issue 5, May 2013, Pages 1240–1246, https://doi.org/10.1093/cercor/bhs122

Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo, Nat Methods, 2004, vol. 1 (pg. 31-37)

R.A. Rocha, J.V. Gimeno-Alcañiz, R. Martín-Ibañez, J.M. Canals, D. Vélez, V. Devesa, Arsenic and fluoride induce neural progenitor cell apoptosis, Toxicology Letters, Volume 203, Issue 3, 2011, Pages 237-244, ISSN 0378-4274, https://doi.org/10.1016/j.toxlet.2011.03.023.

Takata N, Hirase H. Cortical layer 1 and layer 2/3 astrocytes exhibit distinct calcium dynamics in vivo., PLoS ONE, 2008, vol. 3 pg. e2525

Volterra, Andrea, Nicolas Liaudet, and Iaroslav Savtchouk. "Astrocyte Ca2+ signalling: an unexpected complexity." Nature Reviews Neuroscience 15.5 (2014): 327-335.

Yano, S., Tokumitsu, H. & Soderling, T. R. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396, 584–587 (1998).

Yuan Y, Jiang C-y, Xu H, Sun Y, Hu F-f, Bian J-c, et al. (2013) Cadmium-Induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture Is Mediated by a Calcium Signaling Pathway. PLoS ONE 8(5): e64330. https://doi.org/10.1371/journal.pone.0064330

Event: 177: N/A, Mitochondrial dysfunction 1

Short Name: N/A, Mitochondrial dysfunction 1

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Evidence for Perturbation by Stressor

Uranium

Shaki et al. (2012) found that uranyl acetate (UA) exposure in isolated rat kidney mitochondria decreased the ATP production levels and ATP/ADP ratio in a concentration-dependent manner, through inhibition of complexes II and III of the ETC. Both of these levels were significantly changed at UA concentrations of 100 µM and 200 µM. In addition, a concentration-dependent decrease in activity of complex II with exposure to uranium (U) was observed (Shaki et al., 2012). They also found that mitochondrial membrane potential damage and mitochondrial swelling significantly increased both time- and dose-dependently in the treated rat kidneys (Shaki et al., 2012). ATP/ADP ratios were also decreased significantly by treatment with 100 µM or more of uranium (Shaki et al., 2012). Mitochondrial outer membrane damage was significantly decreased by treatment with 200 µM of uranium (Shaki et al., 2012).

Shaki et al. (2013) also investigated the effects of uranium on rat kidneys. They found that mitochondrial permeability transition was also impacted by uranium treatment, causing increased mitochondrial swelling and increased disruption of energy homeostasis (Shaki et al., 2013).

Hao et al. (2014) assessed the changes in mitochondrial potential in human kidney proximal tubular cells treated with uranium and found that the group treated with 500 µM of depleted uranium for 24 hours showed a significant decrease in mitochondrial membrane potential.

In their study of the effects of depleted uranium treatment on human embryonic kidney cells, Hao et al. (2016) found that ETHE1, a mitochondrial protein involved in mitochondrial homeostasis and mitochondrial diseases, had significant dose- and time-dependant decreases in gene expression when treated with 125 µM or more depleted uranium (DU) for 2 hours or more.

Nanoparticles and Micrometer Particles

Karlsson et al. (2009) conducted experiments to examine the effects of micrometer and nanoparticle treatments of copper and iron on human alveolar type-II epithelial cells. Their results showed that copper oxide micrometer and nanoparticle treatments were able to cause dose-dependant mitochondrial depolarization with doses as low as 5 µg/cm² (Karlsson et al., 2009). Iron(III) oxide nanoparticles and micrometer particles were both able to cause similar amounts of mitochondrial depolarization, along with iron (IV) oxide micrometer particles, however they were all much less toxic than copper oxide nanoparticles or micrometer particles (Karlsson et al., 2009).

The effects of gold nanoparticle (Au1.4MS) treatment on human cervical cancer cells were assessed by Pan et al. (2009), who found that the treated cells experienced a significant increase in permeability transition.

Huerta-García et al. (2014) studied the effects of titanium oxide nanoparticle treatment on glial tumor rat neuronal cells and cancerous human brain cells. Their results showed that in the treated rat and human cells there was a clear time-dependant increase in depolarization (Huerta-García et al., 2014). Both the human and rat cells showed time-dependant decreases in mitochondrial membrane potential. The TiO₂ nanoparticles were more toxic to the human cells than to the rat cells. The human cells showed a significant decrease in mitochondrial membrane potential as early as 2 hours post-treatment, while the rat cells did not show significant decrease until 6 hours post-treatment (Huerta-García et al., 2014).

Zhang et al. (2018) investigated the effects of copper nanoparticles on mitochondrial membrane potential in pig kidney cells and found that the treated cells showed a dose-dependant increase in the rate of mitochondrial membrane potential change from 40 µg/mL to 80 µg/mL when treated for 12 hours.

Cadmium

Belyaeva et al. (2012) studied the effects of cadmium treatment on rat kidney cells. In particular, they looked at different respiration rates in treated and untreated rat kidney cell lines. They found that resting respiration rates were significantly stimulated at 48 hours of treatment with 100 µM of cadmium, while uncoupled respiration was unaffected, and basal respiration was enhanced (Belyaeva et al., 2012). These changes in respiration imply that cadmium was capable of reducing the uncoupling efficiency of the cells at concentrations of 100 µM or higher.

Miccadei and Floridi (1993) studied changes in oxygen consumption in rat liver mitochondria which had been treated with cadmium. Their results showed that the treated rats showed a dose-dependant decrease in oxygen consumption which began with doses as low as 3 µM (Miccadei and Floridi, 1993).

Wang et al. (2009), found that, when applied together, lead and cadmium showed individual inhibition and additive effects of rat kidney mitochondrial COX gene expression.

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).

Key Event Description

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.

Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).

Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.

A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).

Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.

How it is Measured or Detected

Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.

I. Mitochondrial dysfunction assays assessing a loss-of function.

1. Cellular oxygen consumption.

See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O₂ consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).

2. Mitochondrial membrane potential (Δψm ).

The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).

3. Enzymatic activity of the electron transport system (ETS).

Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).

4. ATP content.

For the evaluation of ATP levels, various commercially-available ATP assay kits are offered  based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).

II. Mitochondrial dysfunction assays assessing a gain-of function.

1. Mitochondrial permeability transition pore opening (PTP).

The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).

2. mtDNA damage as a biomarker of mitochondrial dysfunction.

Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).

3. Generation of ROS and resultant oxidative stress.

a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.

b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.

c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.

d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (http://www.biotek.com/resources/articles/reactive-oxygen-species.html). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.

e. Detection of hydrogen peroxide (H₂O₂) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H₂O₂ form increasing amounts of fluorescent product (Tarpley et al., 2004).

Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE Oxidative Stress, Increase

Assay Type & Measured Content	Description	Dose Range Studied	Assay Characteristics (Length/Ease of use/Accuracy)
Rhodamine 123 Assay Measuring Mitochondrial membrane potential (MMP) and its collapse  (Shaki et al., 2012)	Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively.	50, 100 and 500 μM of uranyl acetate	Short / easy Medium accurancy
TMRE fluorescence Assay Measuring Mitochondrial permeability transition pore (mPTP) opening (Huser et al., 1998)	Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.	1 µM cyclosporin A	Short / easy Low accurancy
GSH / GSSG Determination Assay Measuring  cellular glutathione (GSH) status; ratio of GSH/GSSG (Owen & Butterfield, 2010; Shaki et al., 2013)	GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.	100 µM uranyl acetate	Short / easy Low accurancy
TBARS Assay Quantification of lipid peroxidation (Yuan et al., 2016)	MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.	200, 400, 800 µM uranyl acetate	Medium / medium High accurancy
Aequorin-based bioluminescence assay Increase in mitochondrial Ca2+ influx (Pozzan & Rudolf, 2009)	Together with GFP, the aequorin moiety acts as Ca2+ sensor in vivo, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.		Short / easy Low accurancy
Western blot & immunostaining analyses Measuring cytochrome c release (Chen et al., 2000)	Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)		Short / easy Medium accurancy
Quantikine Rat/Mouse Cytochrome c Immunoassay Measuring cytochrome c release (Shaki et al., 2012)	Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.		Short / easy Low accurancy
Membrane potential and cell viability – Flow Cytometry Measuring cytochrome c release (Kruidering et al., 1997)	“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 g. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of 60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water et al., 1993)”		Short / easy Medium accurancy

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Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466

Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999, 76:725-734.

Pozzan, T., & Rudolf, R. (2009). Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:https://doi.org/10.1016/j.bbabio.2008.11.012

Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI.

Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379.

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Sanders LH, McCoy J, Hu X, Mastroberardino PG, Dickinson BC, Chang CJ, Chu CT, Van Houten B, Greenamyre JT. (2014a). Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis. 70:214-23.

Sanders LH, Howlett EH2, McCoy J, Greenamyre JT. (2014b) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci. Dec;142(2):395-402.

Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015

Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b

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Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H. (2010) Mouse models in neurological disorders: Applications of non-invasive imaging, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1802, Issue 10, Pages 819-839.

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Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1

Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402.

Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254.

Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44.

Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B

Zhang, H., Chang, Z., Mehmood, K., Abbas, R. Z., Nabi, F., Rehman, M. U., . . . Zhou, D. (2018). Nano copper induces apoptosis in PK-15 cells via a mitochondria-mediated pathway. Biological Trace Element Research, 181(1), 62-70. doi:10.1007/s12011-017-1024-0

Zhou, M., Z.Diwu, Panchuk-Voloshina, N. and R.P. Haughland (1997), A Stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: application in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem 253:162-168.

Event: 1115: Increased, Reactive oxygen species

Short Name: Increased, Reactive oxygen species

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

ROS is a normal constituent found in all organisms.

Key Event Description

Biological State: increased reactive oxygen species (ROS)

Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.

Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). 
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). 

How it is Measured or Detected

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.

Yuan, Yan, et al., (2013) described ROS monitoring by using H₂-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H₂-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.

Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).

Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.

 

References

B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534

Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.

Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.

Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.

Chattopadhyay, Sukumar, et al. "Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants." Toxicology letters 136.1 (2002): 65-76.

Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.

Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.

Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 2016. “Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.” Circulation Research 119 (5): e39–75.

Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.

Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.

Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.

Yen, Cheng Chien, et al. "Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway." Archives of toxicology 85 (2011): 565-575.

Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.

Event: 1262: Apoptosis

Short Name: Apoptosis

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

・Apoptosis is induced in human prostate cancer cell lines (Homo sapiens) [Parajuli et al., 2014].

・Apoptosis occurs in B6C3F1 mouse (Mus musculus) [Elmore, 2007].

・Apoptosis occurs in Sprague-Dawley rat (Rattus norvegicus) [Elmore, 2007].

・Apoptosis occurs in the nematode (Caenorhabditis elegans) [Elmore, 2007].

• Apoptosis occurs in breast cancer cells, human and mouse (Parton)

 

 

Key Event Description

Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1^-/- ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].

Apoptosis is defined as a programmed cell death.  A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe).  Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell.

The Bcl-2 is a protein family suppressing apoptosis by binding and inhibiting two proapoptotic proteins (Bax and Bak) and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases proapoptotic signaling proteins, such as cytochrome c which activated the caspase system. An increased expression of these antiapoptotic proteins (Bcl-2, Bcl-x_L) occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the loss of TP53 tumor suppressor function, or the increase of survival signals (Igf1/2), or decrease of proapoptotic factors (Bax, Bim, Puma) can also increase tumor growth (Hanahan, Juntilla).

In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, Williams, Shahbandi).

How it is Measured or Detected

Apoptosis is characterized by many morphological and biochemical changes such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003].

・DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003].

・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli et al., 2014].

・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli et al., 2014].

・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli et al., 2014].

・Cleavage of PARP is detected with Western blotting [Parajuli et al., 2014].

・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu et al., 2016].

・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].

・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].

・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008]

References

Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283

Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516

Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163

Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257

Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556

Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004

Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313

Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299

Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43

Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831

Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143

Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052

Yasuhara, S. et al. (2003), "Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis", J Histochem Cytochem 51:873-885

Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181

 

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230

Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.

Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). https://doi.org/10.1038/nature03098

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.

Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.

Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230

Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.

Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). https://doi.org/10.1038/nature03098

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.

Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.

Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.

 

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Event: 352: N/A, Neurodegeneration

Short Name: N/A, Neurodegeneration

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The necrotic and apoptotic cell death pathways are quite well conserved throughout taxa (Blackstone and Green, 1999, Aravind et al., 2001). It has been widely suggested that apoptosis is also conserved in metazoans, although despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis (Baum et al., 2007; Blackstone and Green, 1999).

Key Event Description

The term neurodegeneration is a combination of two words - "neuro," referring to nerve cells and "degeneration," referring to progressive damage. The term "neurodegeneration" can be applied to several conditions that result in the loss of nerve structure and function, and neuronal loss by necrosis and/or apoptosis

Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of “neurodegenerative diseases" including Huntington's, Alzheimer’s and Parkinson’s disease. All of these conditions lead to progressive brain damage and neurodegeneration.

Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions, with gross atrophy of the affected regions; symptoms include memory loss.

Parkinson's disease (PD) results from the death of dopaminergic neurons in the midbrain substantia nigra pars compacta; symptoms include bradykinesia, rigidity, and resting tremor.

Several observations suggest correlative links between environmental exposure and neurodegenerative diseases, but only few suggest causative links:

Only an extremely small proportion (less than 5%) of neurodegenerative diseases are caused by genetic mutations (Narayan and Dragounov, 2017). The remainders are thought to be caused by the following:

·      A build up of toxic proteins in the brain (Evin et al., 2006)

·      A loss of mitochondrial function that leads to the oxidative stress and creation of neurotoxic molecules that trigger cell death (apoptotic, necrotic or autophagy) (Cobley et al., 2018)

·      Changes in the levels and activities of neurotrophic factors (Kazim and Iqbal, 2016; Machado et al., 2016; Rodriguez et al., 2014)

·      Variations in the activity of neural networks (Greicius and Kimmel, 2012)

Protein aggregation: the correlation between neurodegenerative disease and protein aggregation in the brain has long been recognised, but a causal relationship has not been unequivocally established (Lansbury et al., 2006; Kumar et al., 2016). The dynamic nature of protein aggregation mean that, despite progress in understanding its mechanisms, its relationship to disease is difficult to determine in the laboratory.

Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered pre-symptomatically, drastically reduce the incidence of these diseases.

Loss of mitochondrial function: many lines of evidence suggest that mitochondria have a central role in neurodegenerative diseases (Lin and Beal, 2006). Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Dysfunction of mitochondria induces oxidative stress, production of free radicals, calcium overload, and mutations in mitochondrial DNA that contribute to neurodegenerative diseases. In all major examples of these diseases there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Moreover, an impressive number of disease- specific proteins interact with mitochondria. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise.

Decreased level of neurotrophic factors: decreased levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer disease and Parkinson disease (Zuccato and Cattaneo, 2009). These studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. Therefore BDNF treatment is being considered as a beneficial and feasible therapeutic approach in the clinic.

Variations in the activity of neural networks: Patients with various neurodegenerative disorders show remarkable fluctuations in neurological functions, even during the same day (Palop et al., 2006). These fluctuations cannot be caused by sudden loss or gain of nerve cells. Instead, it is likely that they reflect variations in the activity of neural networks and, perhaps, chronic intoxication by abnormal proteins that the brain is only temporarily able to overcome.

Neurodegeneration in relation to COVID19

SARS-CoV-2 patients present elevated plasma levels of neurofilament light chain protein (NfL), which is a well-known biochemical indicator of neuronal injury (Kanberg et al., 2020). Postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination (Archie and Cucullo 2020; Heneka et al. 2020).

Autopsy results of patients with SARS showed ischemic neuronal damage and demyelination; viral RNA was detected in brain tissue, particularly accumulating in and around the hippocampus (Gu et al. 2005).

Brain magnetic resonance imaging (MRI) investigations in SARS-CoV-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (FLAIR), along with intracerebral hemorrhagic and microhemorrhagic lesions, and leptomeningeal enhancement (Kandemirli et al. 2020; Kremer et al. 2020; Mohammadi et al., 2020).

Moreover, eight COVID-19 patients with signs of encephalopathy had anti–SARS-CoV-2 antibodies in their CSF, and 4 patients had CSF positive for 14-3-3-protein suggesting ongoing neurodegeneration (Alexopoulos et al. 2020).

How it is Measured or Detected

The assays for measurements of necrotic or apoptotic cell death are described in the Key Event: Cell injury/Cell death

Recent neuropathological studies have shown that Fluoro-Jade, an anionic fluorescent dye, is a good marker of degenerating neurons. Fluoro-Jade and Fluoro-Jade B were found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death (Schmued et al., 2005). More recently, Fluoro-Jade C was shown to be highly resistant to fading and compatible with virtually all histological processing and staining protocols (Schmued et al., 2005). In addition, Fluoro-Jade C is a good tool for detecting acutely and chronically degenerating neurons (Ehara and Ueda, 2009).

Regulatory Significance of the AO

Currently the four available OECD Test Guidelines (TGs) for neurotoxicity testing are entirely based on in vivo neurotoxicity studies: (1)Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (TG 418); (2) Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (TG 419); (3) Neurotoxicity Study in Rodents (TG 424) involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer); (4) Developmental Neurotoxicity (DNT) Study (TG 426) evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. One of the endpoints required by all four of these OECD TGs is evaluation of neurodegeneration that, so far, is performed through in vivo neuropathological and histological studies. Therefore, neurodegeneration described in this AOP as a key event, has a regulatory relevance and could be performed using in vitro assays that allow a reliable evaluation of neurodegeneration using a large range of existing assays, specific for apoptosis, necrosis and autophagy ( see also KE Cell injury/Cell death).

References

Aravind, L., Dixit, V. M., and Koonin, E. V. (2001). Apoptotic Molecular Machinery: Vastly Increased Complexity in Vertebrates Revealed by Genome Comparisons. Science 291, 1279-1284.

Baum, J. S., Arama, E., Steller, H., and McCall, K. (2007). The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14, 1508-1517.

Blackstone, N. W., and Green, D. R. (1999). The evolution of a mechanism of cell suicide. Bioessays 21, 84-88.

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COVID19-related references relevant to KE Neurodegeneration:

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List of Adverse Outcomes in this AOP

Event: 341: Impairment, Learning and memory

Short Name: Impairment, Learning and memory

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).

Life stage applicability: This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). 

Sex applicability: This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). 

Evidence for perturbation by a prototypic stressor: Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). 

Key Event Description

 

Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.

The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).

Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).

For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.

How it is Measured or Detected

In laboratory animals: in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, Hebb-Williams maze, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows.

1) RAM, Barnes, MWM, Hebb-Williams maze are examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004).

2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).

3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).

4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001).

5) Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). 

In humans:  A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and  long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include:

1) Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).

2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).

3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).

4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).

5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).

6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).

7) Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015).

8. Comprehensive developmental inventory for infants and toddlers (CDIIT).  The CDIIT was designed and standardized in 1996, and it measures the global, cognitive, language, motor, gross motor, fine motor, social, self-help and behavioral developmental status of children from 3 to 71 months old (Wang et al., 1998).

In Honey Bees: For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)."

Regulatory Significance of the AO

A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD TG 426) as well as OECD TG 443 (OECD, 2018) both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behaviour.  These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009).

Also, in the frame of the OECD GD 43 (2008) on reproductive toxicity, learning and memory testing may have potential to be applied in the context of developmental neurotoxicity studies. However, many of the learning and memory tasks used in guideline studies may not readily detect subtle impairments in cognitive function associated with modest degrees of developmental thyroid disruption (Gilbert et al., 2012).

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Appendix 2

List of Key Event Relationships in the AOP