AOP ID and Title:

Short Title: SDH inhibition, prolyl hydroxylase inhibition, pseudohypoxia

Authors

Arnaud Tête

Judith favier

Xavier Coumoul

Karine Audouze

Sylvie Bortoli

Status

Coaches

• Rex FitzGerald

Abstract

Succinate dehydrogenase (SDH) is a key enzymatic complex involved in two interconnected metabolic processes for energy production: the transfer of electrons in the mitochondrial respiratory chain and the oxidation of succinate to fumarate in the Krebs cycle. In humans, inherited SDH deficiencies may cause major pathologies including cancers. The cellular and molecular mechanisms related to genetic SDH inactivation have been well described in neuroendocrine tumors, in which it induces an oxidative stress, a pseudohypoxic phenotype, a metabolic, epigenetic and transcriptomic remodeling, and alterations in the migration and invasion capacities of cancer cells, in connection with the accumulation of succinate, an oncometabolite, substrate of the SDH. SDH complex is the molecular target of Succinate Dehydrogenase Inhibitors (SDHi), a family of pesticides widely used to limit the proliferation of pathogenic fungi. This AOP aims to describe the relationship between SDH inactivation and cancer development.

Context

Strategy

Summary of the AOP

Events

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

Key Event Relationships

Stressors

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

References

Appendix 1

List of MIEs in this AOP

Event: 2118: Succinate dehydrogenase, inhibited

Short Name: SDH, inhibited

Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

SDH inhibition by phthalate esters has been measured and quantified in mitochondria of hepatocytes of adult male CD rats (Melnick and Schiller, 1982; Melnick and Schiller, 1985). Inter-species differences in SDH structure may lead to different susceptibilities in different taxa.

SDH inhibition has been demonstrated by lonidamine, 3-nitroproprionic acid (3-NPA) and 2-thenoyltrifluoroacetone (TTFA) in DB-1, HepG2, HCT116 and HeLa cells, and by lonidamine in mitochondria isolated from adult mouse liver (Guo et al, 2016).

Key Event Description

Eukaryotic succinate dehydrogenase (SDH, EC1.3.5.1 (Brenda, IntEnz)) is an enzyme complex comprising four polypeptide chains (SDHA - SDHD) with associated FAD,  Fe-S and haem prosthetic groups that catalyses the reversible oxidation (dehydrogenation) of succinate to fumarate with concomitant reduction of ubiquinone to ubiquinol, serving to channel reducing equivalents from succinate, a tricarboxylic acid (TCA) cycle intermediate, to ubiquinol, an intermediate of the mitochondrial electron transfer chain (Du et al, 2023).

The overall reaction:

succinate + ubiquinone = fumarate + ubiquinol

comprises two, reversible half-reactions:

(1) succinate + FAD = fumarate + FADH₂

and:

(2) FADH₂ + ubiquinone = FAD + ubiquinol

each of which is catalysed at a different active site.

The active site of reaction 1 is in the hydrophilic protein SDHA that contains the covalently bound FAD group, and protudes from the inner mitochondrial membrane (IMM) into the mitochondrial matrix, making it available to exchange succinate and fumarate within the TCA cycle. The active site of reaction 2 is in a more hydrophobic region comprising transmembrane domains of proteins SDHC and SCHD that insert complex II into the IMM (Du et al, 2023), making it available to ubiquinol and ubiquinone shuttling within the IMM.

The presence of two distinct and different active sites enables SDH inibition to be effected in at least two ways: by inhibition of either active site, with potentially different biochemical and physiological consequences, and by inhibitors with differing characteristics.

Inhibition of SDH can result in reduction of mitochondrial electron transport, and subsequent inhibition of oxidative phosphorylation (e.g. Chen et al, 2021), and also generation of superoxide in the mitochondria, leading to with subsequently deleterious effects such as initiation of apoptosis or necrosis (Murphy et al, 2009).

How it is Measured or Detected

Succinate dehydrogenase activity is generally measured by the spectrophotometric detection of colour change in the presence of an  electron acceptor, with succinate (succinic acid) as substrate. Alteration in rate of colour change in the presence of a putative inhibitor determining the strength of that inhibition. The fact that the overall reaction is comprised of two consecutive sub-reactions enables the rate of each sub-reaction - and their inhibition - to be measured separately by appropriate choice of electron acceptor in the presence of succinate as a substrate (e.g. Miyadera et al, 2003). Activities are frequently measured in isolated mitochondria, in order to reduce interference by extra-cytosolic enaymes and intermediates; mitochondria can be sonicated to obviate rate limitation by mitochondrial upake of succinate (e.g. Guo et al, 2016).

SDH activity

Succinate dehydrogenase (SDH) activity corresponds to reaction (1), above. It can be measured by use of the water-soluble dye 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) in the presence of the intermediate electron carrier phenazine methosulfate (PMS), to intercept electrons before their transport to ubiquinone, and convey them to MTT, which changes colour following its reduction.
 

SQR activity

Succinate quinone reductase (SQR) activity corresponds to the overall reaction (i.e. 1 and 2), above. It can be measured by reduction of 2,6-dichlorophenolindophenol (DCPIP) in the presence of the 2,3-dimethoxy-6-methyl-1,4-benzoquinone (UQ₂), which accepts electrons from the ubiquinone reduction site and transfers them to DCPIP, thus being a measure of the rate of the entire reaction catalysed by complex II.

References

Brenda, "Information on EC 1.3.5.1 - succinate dehydrogenase", https://www.brenda-enzymes.org/enzyme.php?ecno=1.3.5.1, accessed 28/04/2023.

Chen, L. et al (2021) "Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation", Journal of Biological Chemistry, Vol 296, 100515

Du, Z. et al (2023) "Structure of the human respiratory complex II", Proceedings of the National Academy of Sciences", Vol 120, e2216713120.

Guo, L. et al (2016) "Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine", Journal of Biological Chemistry, Vol 291. pp42-57.

IntEnz, "IntEnz Enzyme Nomenclature, EC 1.3.5.1", https://www.ebi.ac.uk/intenz/query?cmd=SearchID&id=1525&view=INTENZ, accessed 28/04/2023.

Melnick, R.L. and Schiller, C.M. (1982), "Mitochondrial toxicity of phthalate esters", Environmental Healh Perspectives, Vol 45, pp51-56.

Melnick, R.L. and Schiller, C.M. (1985), "Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria", Toxicology, Vol 34, pp13-27.

Miyadera, H. et al (2003) "Atpenins, potent and specific inhibitors of mitochondrial complex II (succinateubiquinone oxidoreductase)", Proceedings of the National Academy of Sciences, Vol 100, pp473-477.

Murphy, M.P. (2009), "How mitochondria produce reactive oxygen species", Biochemical Journal, Vol 417, pp1-13.

List of Key Events in the AOP

Event: 2243: Succinate Accumulation

Short Name: Succinate Accumulation

AOPs Including This Key Event

Biological Context

Event: 798: Inhibition, Prolyl hydroxylases

Short Name: Inhibition, Prolyl hydroxylases

Event Component

AOPs Including This Key Event

Biological Context

Event: 590: N/A, hypoxia

Short Name: N/A, hypoxia

Event Component

AOPs Including This Key Event

Biological Context

List of Adverse Outcomes in this AOP

Event: 885: Increase, Cancer

Short Name: Increase, Cancer

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