AOP ID and Title:

Short Title: ROS formation leads to cancer via PPAR pathway

Authors

Of the originating work:

Jaesong Jeong and Jinhee Choi, School of Environmental Engineering, University of Seoul, Seoul, Republic of Korea

Of the content populated in the AOP-Wiki:

Daniel L. Villeneuve, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN

Travis Karschnik and John R. Frisch, General Dynamics Information Technology, Duluth, Minnesota

Status

Abstract

Reactive oxygen species (ROS) are derived from oxygen molecules and can occur as free radicals (ex. superoxide, hydroxyl, peroxyl) or non-radicals (ex. ozone, singlet oxygen).  ROS production occurs via a variety of normal cellular process; however, in stress situations (ex. exposure to radiation, chemical or biological stressors) reactive oxygen species levels dramatically increase and cause damage to cellular components.  In this Adverse Outcome Pathway (AOP) we focus on the Peroxisome proliferation-activated receptor (PPAR) response to increases in oxidative stress.  Changes in activation rate of Peroxisome proliferation-activated receptors alter lipid metabolism, and decrease suppression of apoptosis.  In this AOP we focus on the apoptosis response to cellular damage.  Pathways leading to apoptosis, or single cell death, have traditionally been studied as both independent and simultaneous from pathways leading to necrosis, or tissue-wide cell death, with both overlap and distinct mechanisms (Elmore 2007). For the purposes of this AOP, we are characterizing cancer due to widespread cell-death, and recognize the complications in separating the related apoptosis and necrosis pathways.

Background

This Adverse Outcome Pathway focuses on the key pathways in which an established molecular disruption, increased levels of reactive oxygen species (ROS), leads to increased cancer.

Summary of the AOP

Events

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

Key Event Relationships

Overall Assessment of the AOP

1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship  between KEup and KEdown consistent with established biological knowledge?
Key Event Relationship (KER)	Evidence Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.
Relationship 3092: Increased, Reactive oxygen species leads to Decreased, PPAR-gamma activation	Strong support.  Increases in reactive oxygen species (ROS) have been shown to cause a variety of cellular responses including decreased PPARgamma gene expression.
Relationship 3093: Decreased, PPAR-gamma activation leads to Alteration, lipid metabolism	Strong support. Decreased PPAR gene expression have been shown to cause an alteration of lipid metabolism.  PPAR-gamma acts as a nuclear signaling element that controls the transcription of a variety of genes involved in lipid catabolism and energy production pathways.
Relationship 3094: Alteration, lipid metabolism leads to General Apoptosis	Strong support. Alteration of lipid metabolism have been shown to results in abnormal cell function and activity, leading to apoptosis.  Alteration of lipid metabolism leads to changes in cell lipid levels, structural changes in membranes as lipids are key components, and changes in signaling pathways affecting gene and protein expression.  Loss of plasma membrane integrity due to disruptions to lipid metabolism results in cellular processes identifying cells as damaged, which acts as a signal for apoptosis.
Relationship 2977: General Apoptosis leads to Increase, Cancer	Strong support.  The relationship between failure of apoptosis pathways to initiate cell death pathways and increases in cancer is broadly accepted and consistently supported across taxa.
Overall	Strong support.  Extensive understanding of the relationships between events from empirical studies from a variety of taxa.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life Stage: The life stage applicable is all life stages. 

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), telost fish, and invertebrates (cladocerans, mussels).

.

Essentiality of the Key Events

Support for the essentiality of the key events can be obtained from a wide diversity of taxonomic groups, with mammals (lab ice, lab rats, human cell lines), telost fish, and invertebrates (cladocerans and mussels) particularly well-studied.

2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Evidence Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events.   Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events.
MIE 1115: Increased, Reactive oxygen species	Strong support. Increased Reactive oxygen species (ROS) levels are a primary cause of decreases in PPARgamma gene expression.  Evidence is available from studies of stressor exposure and resulting changes in gene expression and protein/enzyme levels.
KE 233: Decreased, PPAR-gamma activation	Strong support. The PPARgamma gene family is important in controlling rate of lipid metabolism.  Evidence is available from studies of stressor exposure and resulting changes in gene expression and protein/enzyme levels.
KE 1060: Alteration, lipid metabolism	Strong support.  Altered lipid metabolism, particularly resulting loss of plasma membrane integrity is a cause of apoptosis.  Evidence is available from studies of stressor exposure and resulting changes in gene expression and protein/enzyme levels.
KE 1513: General Apoptosis	Moderate support. Failure of apoptosis allows cancer cells to proliferate.  Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.
AO 885: Increase, Cancer	Strong support. Cancer proliferates due to a variety of stressors and breakdown of multiple celluar processes.  Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.
Overall	Moderate to strong support.  Direct evidence from empirical studies for most key events, with more inferential evidence rather than direct evidence for apoptosis.

Weight of Evidence Summary

Path	Support
Increased, Reactive oxygen species leads to Decreased, PPAR-gamma activation	Biological plausibility is high.  Representative studies have been done with mammals (El Midaoui et al. 2006; Blanquicett et al. 2010; Lu et al. 2018; Jeong and Choi 2020) fish (Wang et al. 2022).
Decreased, Decreased, PPAR-gamma activation leads to Alteration, lipid metabolism	Biological plausibility is high.  Representative studies have been done with mammals (Chamorro-Garcia et al. 2018; Jeong and Choi 2020); fish (Venezia et al. 2021).  For review (Tickner et al. 2001; Berger and Moller 2002; Luquet et al. 2005; Den Broeder et al. 2015).
Alteration, lipid metabolism leads to General Apoptosis	Biological plausibility is high.  Representative studies have been done with mammals (Cadet et al. 2010, Gao et al. 2020); invertebrates (Avio et al. 2015). For review (Huang and Freter 2015).
General Apoptosis leads to Increase, Cancer	Biological plausibility is high.  Representative studies have been done with mammals (Pavet et al. 2014; Jeong and Choi 2020).  For review (Heinlein and Chang 2004; Vihervaara and Sistonen 2014).

3. Empirical Support for Key Event Relationship: Does empirical evidence support that a  change in KEup leads to an appropriate change in KEdown?
Key Event Relationship (KER)	Evidence Strong =  Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.
Relationship 3092: Increased, Reactive oxygen species leads to Decreased, PPAR-gamma activation	Strong support. Increases in ROS leads to decreases in PPAR gamma gene expression, primarily by examining gene expression levels.
Relationship 3093: Decreased, PPAR-gamma activation leads to Alteration, lipid metabolism	Strong support. Decreases in PPAR gamma expression leads to alteration of lipid metabolism, primarily by assessing lipid content and levels of energy metabolites.
Relationship 3094: Alteration, lipid metabolism leads to General Apoptosis	Strong support. Altered lipid metabolism leads to apoptosis; problems with lipid metabolism lead to abnormal cells, triggering apoptosis pathways.
Relationship 2977: General Apoptosis leads to Increase, Cancer	Strong support. Mechanistic studies show that failure for apoptosis to eliminate cancer cells allows increases in cancer proliferation.
Overall	Strong support. Exposure from empirical studies shows consistent change in both events from a variety of taxa.

For overview of the biological mechanisms involved in this AOP, see Liu et al. (2015) and Jeong and Choi (2020); their studies analyzed ToxCast in vitro assays of mammalian acute toxicity data to identify correlations between toxicity pathways and chemical stressors, providing support for the key event relationships represented here.

References

Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., D’Errico, G., Pauletto, M., Bargelloni, L., and Regoli, F.  2015.  Pollutants bioavailability and toxicological risk from microplastics to marine mussels.  Environmental Pollutants 198: 211-222.

Berger, J. and Moller, D.  2002.  The mechanisms of action of PPARS.  Annual Review of Medicine 53: 409-435.

Blanquicett, C., Kang, B-Y., Ritzenthaler, J.D. Jones, D.P., and Hart, C.M.  2010.  Free Radical Biology and Medicine 48: 1618-1625.

Den Broeder, M.J., Kopylova, V.A., Kamminga, L.M. Legler, J.  2015.  Zebrafish as a model to study the role of peroxisome proliferating-activated receptors in adipogenesis and obesity.  PPAR Research 2015: 358029.

 

Cadet, J.L., Jayanthi, S., McCoy, M.T., Beauvais, G., and Cai, N.S.  2010.  Dopamine D1 receptors, regulation of gene expression in the brain, and neurogeneration.  CNS Neurological Disorders - Drug Targets 9: 526-538.

Chamorro-Garcia, R., Shoucri, B.M., Willner, S., Kach, H., Janesick, A., and Blumberg, B.  2018.  Effect of perinatal exposure to dibutyltin chloride on fat and glucose metabolism in mice, and molecular mechanisms, in vitro.  Environmental Health Perspectives 126: 057006.

El Midaoui, A., Wu, L., Wang, R., and de Champlain, J.  2006.  Modulation of cardiac and aortic peroxisome proliferator-activated receptor-gamma expression by oxidative stress in chronically glucose-fed rats.  American Journal of Hypertension 19: 407-412.

 

Gao, L., Xu, Z., Huang, Z., Tang, Y., Yang, D., Huang, J., He, L., Liu, M., Chen, Z., and Teng, Y.  2020.  CPI-613 rewires lipid metabolism to enhance pancreatic cancer apoptosis via the AMPK-ACC signaling.  39: 73.

Heinlein, C.A. and Chang, C.  2004.  Androgen receptor in prostate cancer.  Endocrine Reviews 25: 276-308.

Huang, C. and Freter, C.  2015.  Lipid metabolism, apoptosis and cancer therapy.  International Journal of Molecular Sciences 16: 924-949.

 

Jeong, J. and Choi, J.  2019.  Adverse outcome pathways potentially related to hazard identification of microplastics based on toxicity mechanisms. Chemosphere 231: 249-255.

Jeong, J. and Choi, J.  2020.  Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach.  Environment International 137:105557.

Liu, J., Mansouri, K., Judson, R.S., Martin, M.T., Hong, H., Chen, M., Xu, X., Thomas, R.S., and Shah, I.  2015.  Predicting hepatoxicity using ToxCast in vitro bioactivity and chemical structure.  Chemical Research in Toxicology 28: 738-751.

Lu, L., Wan, Z., Luo, T., Fu, Z., and Jin, Y.  2018.  Polystyrene microplastics induce microbiota dysbiosis and hepatic lipid metabolism disorder in mice. Science of the Total Environment 631-632: 449-458.

Luquet, S., Gaudel, C., Holst, D., Lopez-Soriano, J., Jehl-Pietri, C., Fredenrich, A., and Grimaldi, P.A.  2005.  Roles of PPAR delta in lipid absorption and metabolism: A new target for the treatment of type 2 diabetes.  Biochimica and Biophysica Acta 1740: 313-317.

Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H.  2014.  Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells.  Cell Death and Disease 5: e1043.

Tickner, J.A., Schettler, T., Guidotti, T., Mccally, M., and Rossi, M.  2001.  Health risks posed by used of di-2-ethylhexyl phthalate (DEHP) in PVC medical devices: A critical review.  American Journal of Industrial Medicine 39: 100-111.

Venezia, O., Islam, S., Cho, C., Timme-Laragy, A.R., and Sant, K.E.  2021.  Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.  Toxicology and Applied Pharmacology 426: 115653.

Vihervaara, A. and Sistonen, L.  2014.  HSF1 at a glance.  Journal of Cell Scientce 127: 261-266.

Wang, X., Ma, Q., Chen, L. Wu, H., Chen, L.-Q., Qiao, F., Luo, Y., Zhang, M.-L., and Du, Z.-Y.  2022.  Peroxisome proliferator-activated receptor gamma is essential for stress adaptation by maintaining lipid homeostatis in female fish.  Biochimica et Biophysica Acta – Molecular and Cell Biology of Lipids 1867: 159162.

 

 

Appendix 1

List of MIEs in this AOP

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.

List of Key Events in the AOP

Event: 233: Decreased, PPAR-gamma activation

Short Name: Decreased, PPAR-gamma activation

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Life Stage: All life stages. 

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly, with representative studies in mammals.

Key Event Description

The Peroxisome Proliferator-Activated Receptors (PPAR) family of genes involved in regulation of lipid metabolism and energy pathways (Desvergne and Wahli 1999, Hihi et al. 2002, Ahmed et al. 2007).  Fatty acids stimulate the expression of PPAR genes, which initiate a variety of cellular responses focused on lipid metabolism, but also inflammation and apoptosis pathways.  Decreases in PPAR-gamma expression are associated with disruption of adipocyte differentiation and glucose homeostasis.

How it is Measured or Detected

Peroxisome proliferation-activated receptors are investigated by changes in gene expression and protein levels.  X-ray crystallography can be used to determine molecular structure.  Effects of PPAR gamma on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches. 

References

Ahmed, W., Ziouzenkova, O., Brown, J. Devchand, P. Francis, S., Kadakia, M., Kanda, T., Orasanu, G., Sharlach, M., Zandbergen, F., and Plutzky, J.  2007.  PPARs and their metabolic modulation: new mechanisms for transcriptional regulation?  Journal of Internal Medicine 262: 184-198.

Desvergne, B. and Wahli, W. 1999.  Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20(5): 649-688.

Hihi, A.K., Michalik, L., Wahli, W. 2002. PPARs: transcriptional effectors of fatty acids and their derivatives. Cellular and Molecular Life Sciences 59: 790-798.

Event: 1060: Alteration, lipid metabolism

Short Name: Alteration, lipid metabolism

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Life Stage: All life stages. 

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly, with representative studies in mammals.

Key Event Description

Lipids are important molecules for efficient energy storage, in addition to roles as signaling molecules and basic building blocks in organisms.  In addition to energy release, lipid metabolism affects the amount of stored fat.  Alteration of lipid metabolism reflects a disruption of normal function, as evidenced by changes in gene expression, enzyme levels, break-down products, or fat content.  Peroxisome proliferation-activated receptors pathways (and associated genes and proteins) are commonly monitored for downstream effects on lipid metabolism (Luquet et al. 2005; Den Broeder et al. 2015; Chamorro-Garcia et al. 2018; Venezia et al. 2021).

How it is Measured or Detected

Changes in lipid metabolism can be detected by examining organism fat content, or by examination of organs (ex. stomach, liver, intestines) for break-down products (ex. proteins) or changes in gene expression.

References

Chamorro-Garcia, R., Shoucri, B.M., Willner, S., Kach, H., Janesick, A., and Blumberg, B.  2018.  Effect of perinatal exposure to dibutyltin chloride on fat and glucose metabolism in mice, and molecular mechanisms, in vitro.  Environmental Health Perspectives 126: 057006.

Den Broeder, M.J., Kopylova, V.A., Kamminga, L.M. Legler, J.  2015.  Zebrafish as a model to study the role of peroxisome proliferating-activated receptors in adipogenesis and obesity.  PPAR Research 2015: 358029.

Luquet, S., Gaudel, C., Holst, D., Lopez-Soriano, J., Jehl-Pietri, C., Fredenrich, A., and Grimaldi, P.A.  2005.  Roles of PPAR delta in lipid absorption and metabolism: A new target for the treatment of type 2 diabetes.  Biochimica and Biophysica Acta 1740: 313-317.

Venezia, O., Islam, S., Cho, C., Timme-Laragy, A.R., and Sant, K.E.  2021.  Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.  Toxicology and Applied Pharmacology 426: 115653.

Event: 1513: General Apoptosis

Short Name: General Apoptosis

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: appears to be present broadly among multicellular organisms.

Key Event Description

Apoptosis is the programmed cell death in general. This process is well regulated with a sequence of events before cell fragmentation occurs. Changes in the nucleus of a cell are the first step in apoptosis. Before that, other factors such as stress, inflammation, cell damage can induce expression or activation of signal proteins which will activate the pathway for apoptosis. Examples of proteins which are involved in apoptosis are the proteins p53, Bcl-2, JNK, and several caspases. When the first step is taken in the apoptosis process the cell will end in membrane-bounded apoptotic bodies. These bodies are cleared by macrophages or other cells where the degradation process starts within heteorphagosomes.

How it is Measured or Detected

There are several possibilities to measure and detect apoptosis, some common techniques are:

• The detection of Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) substances which are released from cells which undergo apoptosis.
• An older but effective technique it the annexin V – affinity assay. The principle of this assay is the high affinity binding between annexin V and phosphatidylserine. In a vital cell there is a membrane lipid asymmetry where phosphatidylserine molecules are facing the cytosol. During apoptosis the membrane lipid asymmetry is lost, and the phosphatidylserine molecules are expressed in the outer membrane. When annexin-V is present in combination with Ca²⁺ it binds with high affinity to phosphatidylserine. With a hapten label at the annexin-V this process can be detected.
• Another technique is the detection of cleaved caspase-3, which could be done with western blot or enzyme-linked immunosorbent assays.
• Cytochrome c is also a protein which is released in an early stage of apoptosis. Detection of cytochrome c can be done with metal nanoclusters which have a fluorescent probe in addition to western blot assay.

References

Shtilbans, V., Wu, M. & Burstein, D. E. Evaluation of apoptosis in cytologic specimens. Diagnostic Cytopathology 38, 685–697 (2010).

Wu, J., Sun, J. & Xue, Y. Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells. Toxicol. Lett. 199, 269–276 (2010).

Redza-Dutordoir, M. & Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim. Biophys. Acta - Mol. Cell Res. 1863, 2977–2992 (2016).

Lossi, L., Castagna, C. & Merighi, A. Neuronal cell death: An overview of its different forms in central and peripheral neurons. in Neuronal Cell Death: Methods and Protocols 1–18 (2014). doi:10.1007/978-1-4939-2152-2_1

Van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B. & Reutelingsperger, C. P. M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1–9 (1998).

Shamsipur, M., Molaabasi, F., Hosseinkhani, S. & Rahmati, F. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes. Anal. Chem. 88, 2188–2197 (2016).

List of Adverse Outcomes in this AOP

Event: 885: Increase, Cancer

Short Name: Increase, Cancer

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Life Stage: All life stages.  Older individuals are more likely to manifest this key event (adults > juveniles > embryos).

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).

Key Event Description

Cancer is a general key event for related diseases each exhibiting uncontrolled proliferation of abnormal cells (for review see Hanahan and Weinberg 2011).  A cancer often is initially associated with a specific organ, with malignant tumors developing ability to metastasize, or travel to other areas of the body.  Most cancers develop from genetic mutations in normal cells, although a minority of cancers are hereditary.   Exposure to chemical stressors, radiation, tobacco smoke, or viruses can increase the likelihood that cancer will develop.

Cancer cells proliferate due to capabilities summarized by Hanahan and Weinberg (2011):

1. Sustained proliferation signaling – by deregulating normal cell signals, cancer cells can sustain chronic proliferation.
2. Evading growth suppressors – by evading activities of tumor suppressor genes, cancer cells continue to proliferate.
3. Activating invasion and metastasis – by altering shape and attachment to cells in the extracellular matrix, cancer cells gain ability to move to other locations.
4. Enabling replicative immortality – by disabling senescence pathways, cancer cells have extended lifespans.
5. Inducing angiogenesis – by enabling neovasculature, cancer cells receive nutrients and oxygen and get rid of waste products.
6. Resisting cell death – by evading apotosis and necrosis defense pathways, cancer cells avoid elimination.

How it is Measured or Detected

Most carcinogenicity studies are conducted with rodents (see OECD 2018; Zhou et al. 2023 for methods) or in-vitro with mammalian cell lines (see OECD 2023 for methods).  Cancer is usually detected by biopsy or histopathological examination of tissue.  Gene expression levels can also be assessed, as increased transcription of known genes have been associated with specific cancers (ex. Tumor Necrosis Factor (Pavet et al. 2014); Heat Shock Factors (Vihervaara and Sistonen 2014; Androgen Receptor (Heinlein and Chang 2004)).

Regulatory Significance of the AO

Cancer is a critical endpoint in human health risk assessment.   It is embedded in regulatory frameworks for human health protection in many countries (see OSHA 2023 for examples of US regulations and European Parliament 2022 for examples of regulations in Europe).

References

Abraha, A.M. and Ketema, E.B.  2016.  Apoptotic pathways as a therapeutic target for colorectal cancer treatment.  World Journal of Gastrointestinal Oncology 8 (8): 583-491

European Parliament.  2022.  Directive 2004/37/EC of the European Parliament on the protection of workers from the risks related to exposure to carcinogens, mutagens or reprotoxic substances at work.  Retrieved 3 August 2023 from http://data.europa.eu/eli/dir/2004/37/2022-04-05

Hanahan, D. and Weinberg, R.A.  2011.  Hallmarks of cancer: the next generation.  Cell 144(5): 646-674.

Heinlein, C.A. and Chang, C.  2004.  Androgen receptor in prostate cancer.  Endocrine Reviews 25: 276-308.

OECD.  2018.  Test no. 451: OECD Guideline for the Testing of Chemicals: Carcinogenicity Studies.  OECD Publishing, Paris.  Retrieved 3 August 2023 from https://www.oecd.org/env/test-no-451-carcinogenicity-studies-9789264071186-en.htm

OECD.  2023. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.  Retrieved 3 August 2023 from  https://doi.org/10.1787/9789264264861-en.htm

OSHA. 2023.  Carcinogens.  Retrieved 3 August 2023 from https://www.osha.gov/carcinogens/standards

Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H.  2014.  Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells.  Cell Death and Disease 5: e1043.

Vihervaara, A. and Sistonen, L.  2014.  HSF1 at a glance.  Journal of Cell Scientce 127: 261-266.

Zhou, Y., Xia, J., Xu, S., She, T., Zhang, Y., Sun, Y., Wen, M., Jiang, T., Xiong, Y., and Lei, J.  2023.  Experimental mouse models for translational human cancer research.  Frontiers in Immunology 14: 1095388.

Appendix 2

List of Key Event Relationships in the AOP