## 4.2.4. Cytotoxicity: xenobiotic compounds causing cell death

Authors: Frank Van Belleghem, Karen Smeets

Reviewers: Timo Hamers, Bas J. Blaauboer

Learning objectives:

You should be able to:

- name the main factors that cause cell death,
- describe the process of necrosis and apoptosis,
- describe the morphological differences between apoptosis and necrosis,
- explain what form of cell death is caused by chemical substances.

Keywords: cell death, apoptosis, necrosis, caspase activation, mitochondrial permeability transition

Description

Cytotoxicity or cell toxicity is the result of chemical-induced macromolecular damage (see the section on Protein inactivation) or receptor-mediated disturbances (see the section on Receptor interactions). Initial events such as covalent binding to DNA or proteins; loss of calcium control or oxidative stress (see the sections on Oxidative stress I and II) can compromise key cellular functions or trigger cell death. Cell death is the ultimate endpoint of lethal cell injury; and can be caused by chemical compounds, mediator cells (i.e. natural killer cells) or physical/environmental conditions (i.e. radiation, pressure, etc.). The multistep process of cell death involves several regulated processes and checkpoints to be passed before the cell eventually reaches a point of no return, leading to either programmed cell death or apoptosis, or to a more accidental form of cell death, called necrosis. This section describes the cytotoxic process itself, in vitro cytotoxicity testing is dealt with in the section on Human toxicity testing - II. In vitro tests.

Chemical toxicity leading to cell death

Cells can actively maintain the intracellular environment within a narrow range of physiological parameters despite changes in the conditions of the surrounding environment. This internal steady-state is termed cellular homeostasis. Exposure to toxic compounds can compromise homeostasis and lead to injury. Cell injury may be direct (primary) when a toxic substance interacts with one or more target molecules of the cell (e.g. damage to enzymes of the electron transport chain), or indirect (secondary) when a toxic substance disturbs the microenvironment of the cell (e.g. decreased supply of oxygen or nutrients). The injury is called reversible when cells can undergo repair of adaptation to achieve a new viable steady state. When the injury persists or becomes too severe, it becomes irreversible and the cell eventually perishes, thereby terminating cellular functions like respiration, metabolism, growth and proliferation, resulting in cell death (Niesink et al., 1996).

The main factors determining the occurrence of cell death are:

- the nature and concentration of the active toxic compound - in some cases a reactive intermediate - and the availability of that agent at the site of the target molecules;
- the role of the target molecules in the functioning of the cell and/or maintaining the microenvironment;
- the effectiveness of the cellular defence mechanisms in the detoxication and elimination of active agents, in repairing (primary) damage, and in the ability to induce proteins that either promote or inhibit the cell death process.

It is important to realize that also “harmless” substances such as glucose or salt may lead to cell injury and cell death by disrupting the osmotic homeostasis at sufficient concentrations. Even an essential molecule such as oxygen causes cell injury at sufficiently high partial pressures (see the sections on Oxidative stress I and II). Apart from that, all chemicals exert “baseline toxicity” (also called “narcosis”) as described in the textbox “narcosis and membrane damage” in the section on Toxicodynamics & Molecular Interactions.

The main types of cell death: necrosis and apoptosis
The two most important types of cell death are necrosis or accidental cell death (ACD) and apoptosis, a form of programmed cell death (PCD) or cell suicide. Cellular imbalances that initiate or promote cell death alone or in combination are oxidative stress, mitochondrial injury or disturbed calcium fluxes. These alterations are reversible at first, but after progressive injury, result in irreversible cell death. Cell death can also be initiated via receptor-mediated signal transduction processes. Apoptotic and necrotic cells differ in both the morphological appearance as well as biochemical characteristics. Necrosis is associated with cell swelling and a rapid loss of membrane integrity. Apoptotic cells shrink into small apoptotic bodies. Leaking cells during necrosis induce inflammatory responses, although inflammation is not entirely excluded during the apoptotic process (Rock & Kono, 2008).
Necrosis
Necrosis has been termed accidental cell death because it is a pathological response to cellular injury after exposure to severe physical, chemical, or mechanical stressors. Necrosis is an energy-independent process that corresponds with damage to cell membranes and subsequent loss of ion homeostasis (in particular Ca2+). Essentially, the loss of cell membrane integrity allows enzymes to leak out of the lysosomal membranes, destroying the cell from the inside. Necrosis is characterized by swelling of cytoplasm and organelles, rupture of the plasma membrane and chromatin condensation (see Figure 1). These morphological appearances are associated with ATP depletion, defects in protein synthesis, cytoskeletal damage and DNA-damage. Besides, cell organelles and cellular debris leak via the damaged membranes into the extracellular space, leading to activation of the immune system and inflammation (Kumar et al., 2015). In contrast to apoptosis, the fragmentation of DNA is a late event. In a subsequent stage, injury is propagated across the neighbouring tissues via the release of proteolytic and lipolytic enzymes resulting in larger areas of necrotic tissue. Although necrosis is traditionally considered as an uncontrolled form of cell death, emerging evidence points out that the process can also occur in a regulated and genetically controlled manner, termed regulated necrosis (Berghe et al., 2014). Moreover, it can also be an autolytic process of cell disintegration after the apoptotic program is completed in the absence of scavengers (phagocytes), termed post-apoptotic or secondary necrosis (Silva, 2010).
Apoptosis
Apoptosis is a regulated (programmed) physiological process whereby superfluous or potentially harmful cells (for example infected or pre-cancerous cells) are removed in a tightly controlled manner. It is an important process in embryonic development, the immune system and in fact, all living tissues. Apoptotic cells shrink and break into small fragments that are phagocytosed by adjacent cells or macrophages without producing an inflammatory response (Figure 3). It can be seen as a form of cellular suicide because cell death is the result of induction of active processes within the cell itself. Apoptosis is an energy-dependent process (it requires ATP) that involves the activation of caspases (cysteine-aspartyl proteases), pro-apoptotic proteins present as zymogens (i.e. inactive enzyme precursors that are activated by hydrolysis). Once activated, they function as cysteine proteases and activate other caspases. Caspases can be distinguished into two groups, the initiator caspases, which start the process, and the effector caspases, which specifically lyse molecules that are essential for cell survival (Blanco & Blanco 2017). Apoptosis can be triggered by stimuli coming from within the cell (intrinsic pathway) or from the extracellular medium (extrinsic pathway) as shown in Figure 2. The extrinsic pathway activates apoptosis in response to external stimuli, namely by extracellular ligands binding to cell-surface death receptors (Tumour Necrosis Factor Receptor ((TNFR)), leading to the formation of the death-inducing signalling complex (DISC) and the caspase cascade leading to apoptosis. The intrinsic pathway is activated by cell stressors such as DNA damage, lack of growth factors, endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) burden, replication stress, microtubular alterations and mitotic defects (Galluzzi et al., 2018). These cellular events cause the release of cytochrome c and other pro-apoptotic proteins from the mitochondria into the cytosol via the mitochondrial permeability transition (MPT) pore. This is a megachannel in the inner membrane of the mitochondria composed of several protein complexes that facilitate the release of death proteins such as cytochrome c. The opening is triggered and tightly regulated by anti-apoptotic proteins, such as B-cell lymphoma-2 (Bcl-2) and pro-apoptotic proteins, such as Bax (Bcl-2 associated X protein) and Bak (Bcl-2 antagonist killer). The intrinsic and extrinsic pathways are regulated by the apoptosis inhibitor protein (AIP) which directly interacts with caspases and suppresses apoptosis. The release of the death protein cytochrome c induces the formation of a large protein structure formed in the process of apoptosis (the apoptosome complex) activating the caspase cascade leading to apoptosis. Other pro-apoptotic proteins oppose to Bcl (SMAC/Diablo) and stimulate caspase activity by interfering with AIP (HtrA2/Omi). HtrA2/Omi also activates caspases and endonuclease G (responsible for DNA degradation, chromatin condensation, and DNA fragmentation). The apoptosis-inducing factor (AIF) is involved in chromatin condensation and DNA fragmentation. Many xenobiotics interfere with the MPT pore and the fate of a cell depends on the balance between pro- and anti-apoptotic agents (Blanco & Blanco, 2017).

Figure 1. This diagram shows the observable differences between necrotic and apoptotic cell death. Reversible injury is characterized by cytoplasmic enlargement (oncosis), membrane blebbing, swelling of endoplasmic reticula and mitochondria, and the presence of myelin figures (twirled phospholipid masses from damaged cell membranes). Progressive injury leads to the necrotic breakdown of membranes, organelles and the nucleus. The nucleus can thereby undergo shrinking (pyknosis), fragmentation (karyorrhexis) or complete dissolution with loss of chromatin (karyolysis) (see in-set 1). The cell is eventually disrupted, releasing its contents and inducing an inflammatory reaction. In contrast, a cell undergoing apoptosis displays cell shrinkage, membrane blebbing, and (ring-shaped) chromatin condensation (see in-set 2, image adapted from Toné et al., 2007). The nucleus and cytoplasm break up into fragments called apoptotic bodies, which are phagocytosed by surrounding cells or macrophages.

Figure 2. Scheme of the factors involved in the apoptotic process. APAF-1, Apoptosis protease activator factor-1; Bak, Bcl-associated antagonist killer; Bax, Bcl-associated X protein; Bcl-2, B cell lymphoma-2; DD, death domain; DED, death effector domain; IAP, inhibitor apoptosis protein; AIF, apoptosis-inducing factor; TNFR, tumour necrosis factor receptor; TRADD, TNFR-associated death domain. Image adapted from Blanco & Blanco 2017.

What determines the form of cell death caused by chemical substances?
Traditionally, toxic cell death was considered to be uniquely of the necrotic type. The classic example of necrosis is the liver toxicity of carbon tetrachloride (CCl4) caused by the biotransformation of CCl4 to the highly reactive radicals (CCl3• and CCl3OO•).

Several environmental contaminants including heavy metals (Cd, Cu, CH3Hg, Pb), organotin compounds and dithiocarbamates can exert their toxicity via induction of apoptosis, likely mediated by disruption of the intracellular Ca2+ homeostasis, or induction of mild oxidative stress (Orrenius et al., 2011).

In addition, some cytotoxic substances (e.g. arsenic trioxide (As2O3)) tend to induce apoptosis at low exposure levels or early after exposure at high levels, whereas they cause necrosis later at high exposure levels. This implicates that the severity of the insult determines the mode of cell death (Klaassen, 2013). In these cases, both apoptosis and necrosis involve the dysfunction of mitochondria, with a central role for the mitochondrial permeability transition (MPT). Normally, the mitochondrial membrane is impermeable to all solutes except for the ones having speciﬁc transporters. MPT allows the entry into the mitochondria of solutes with a molecular weight of lower than 1500 Daltons, which is caused by the opening of mitochondrial permeability transition pores (MPTP) in the inner mitochondrial membrane. As these small-molecular-mass solutes equilibrate across the internal mitochondria membrane, the mitochondrial membrane potential (ΔΨmt) vanishes (mitochondrial depolarization), leading to uncoupling of oxidative phosphorylation and subsequent adenosine triphosphate (ATP) depletion. Moreover, since proteins remain within the matrix at high concentration, the increasing colloidal osmotic pressure will result in movement of water into the matrix, which causes swelling of the mitochondria and rupture of the outer membrane. This results in the loss of intermembrane components (like cytochrome c, AIF, HtrA2/Omi, SMAC/Diablo & Endonuclease G) to the cytoplasm. When MPT occurs in a few mitochondria, the affected mitochondria are phagocytosed and the cell survives. When more mitochondria are affected, the release of pro-apoptotic compounds will lead to the caspase activation resulting in apoptosis. When all mitochondria are affected, ATP becomes depleted and the cell will eventually undergo necrosis as shown in Figure 3 (Klaassen et al., 2013).

Figure 3. Dose-response relationship of toxicant-induced modes of cell death. The mode of cell death triggered by some toxicants is dose-dependent. Most often, exposure to low doses results in apoptosis, whereas higher levels of the same toxicant might cause necrosis. Image adapted from Klaassen et al., 2013.

References

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Blanco, G., Blanco, A. (2017). Chapter 32 – Apoptosis. Medical biochemistry. (pp. 791-796) Academic Press. https://doi.org/10.1016/B978-0-12-803550-4.00032-X

Galluzzi, L., Vitale, I., Aaronson, S.A., Abrams, J.M., Adam, D., Agostinis, P., ... & Annicchiarico-Petruzzelli, M. (2018). Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death & Differentiation, 1. https://doi.org/10.1038/s41418-017-0012-4

Klaassen, C.D., Casarett, L.J., & Doull, J. (2013). Casarett and Doull's Toxicology: The basic science of poisons (8th ed.). New York: McGraw-Hill Education / Medical.
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Kumar, V., Abbas, A.K.,& Aster, J.C. (2015). Robbins and Cotran pathologic basis of disease, professional edition. Elsevier Health Sciences. ISBN 978-0-323-26616-1.

Niesink, R.J.M., De Vries, J., Hollinger, M.A. (1996) Toxicology: Principles and Applications, (1st ed.). CRC Press. ISBN 0-8493-9232-2.

Orrenius, S., Nicotera, P., Zhivotovsky, B. (2011). Cell death mechanisms and their implications in toxicology. Toxicological Sciences 119, 3-19. https://doi.org/10.1093/toxsci/kfq268

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Silva, M.T. (2010). Secondary necrosis: the natural outcome of the complete apoptotic program. FEBS Letters 584, 4491-4499. https://doi.org/10.1016/j.febslet.2010.10.046

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