Mechanistic paradigms of cell death - revisited

Introduction

Cell was first discovered and described by Robert Hooke in his book "Micrographia" in 1665. Anton van Leeuwenhoek was another scientist who saw the cells in 1676. Cell theory was proposed by two scientists Theodor Schwann and Matthias Jacob Schleiden in 1839. A German Physician Rudolph Ludwig Carl Virchow (1858) further added a third tenet to cell theory that postulated "Omnis cellula e cellula", literally meaning -every cell stems from pre-existing cell. In subsequent years Virchow made cellular pathology a subject of overwhelming importance. He delivered 20 lectures that were published in, "Cellular Pathology as based upon Physiological and Pathological Histology", a classical work that transformed scientific thought of cell biology. Another benchmark publication," Introduction to the Study of Experimental Medicine", by Claude Bernard (1865) introduced the term cellular homeostasis. Thereafter, the concepts of cell injury /cell death were considered fundamental to study the pathogenesis of disease. Several breakthroughs exploded between 1850s and 1950s. These included the discovery ofnucleus (Robert Brown, 1831); mitochondria (Kolliker, 1857), golgi (Golgi,1898); structure of DNA ( Watson and Crick, 1952-53) and endoplasmic reticulum (Keith Porter,1953). American Society of Cell Biology, celebrated 40 years of its inception and published landmark papers in cell biology in 2000. Discovery of immortal HeLa Cells (1951); DNA polymerase (1956); transmission electron microscope (1939) and PCR (1983) influenced research activities on cell, all over the globe. I am a witness to many researches that were focused to find an answer to the question-how cells die? Some of these answers are reported in this article. What little contribution could be made by me and my colleagues to address this question during five decades is also described.

The process of cell death raises basic questions on creation and maintenance of an organism. Cell death is required to create an order in the organism. We do know that cells die in a controlled manner. The role of cell death in embryonic th development has been recognized since 19 century when embryologists recognized the basis of sexual differentiation, formation of complex structures such as limbs and disappearance of vestigial organs. Till (1981) classified cellular events of development in two categories. Lamarckian development-suggests the role of environment in the modification of genome. Whereas Darwinian concept proposes the role of gene expression in life or death of a cell. Both these processes create an order in cellular differentiation and homeostasis in embryonic life. Michaelson (1987) hypothesized that cell death is more a cause than result of developmental organization. Superfluous, irreversibly damaged or potentially harmful cells need to be removed by suitable intrinsic mechanisms (Conrad et al., 2016). Regulated cell death (RCD) occurs not only in multi-cellular organisms but has also been reported in several yeast species, Dictyostelium discoideum and in prokaryotes like Escherichia coli (Cornillon et al. 1998;Eisenberg et al. 2007). In contrast, accidental cell death (ACD) is instantaneous and catastrophic demise of cells that occurs in organisms/systems exposed to physical, chemical and/or mechanical stress. Different types of RCD and ACD can be separated in three types.

Types of cell death : Historically, cell death has been classified on the basis of a spectrum of morphological changes in the cells. Nomenclature Committee on Cell Death (Galluzzi et al., 2018) classified them into three categories-Type I cell death : it includes apoptosis exhibiting cytoplasmic shrinkage, chromatin condensation (pycnosis), nuclear fragmentation (karyorrhexis) and membrane blebbing. Necrosis : Our interest in necrotic cell death perhaps emerged in 1970s. A chapter entitled," Experimental Toxic Injury of the Liver", written by Professor Rouiller who edited " The Liver, Morphology, Biochemistry and Physiology, Part I and II (Academic Press London and New York) introduced this term necrosis. It has been derived from a Greek word ,"nekrosis" meaning cell injury that results in premature death of the cell due to autolysis. It was realized that different pathological conditions viz. hypoxia, trauma, exposure to toxins, ischemia, viral or bacterial infection and neurodegenerative disorders can induce necrotic cell death (Fig. 1). Morphologically, necrosis can be classified as coagulative necrosis, liquefactive necrosis, caseous necrosis, fat necrosis and fibrinoid necrosis. Pathogenesis of cell injury may be sublethal with chances of recovery or cell may pass through a phase of "no return". Phase of reversible injury exhibits hydropic degeneration, cloudy swelling or vacuolar degeneration (Rouiller, 1964;Popper and Schaffner, 1957;Arias et al., 1988;Zimmerman, 1978). In the late stage of necrosis, cytoplasm loses contents and exhibits homogeneous eosinophilic appearance, membrane changes, mitochondrial swelling, formation of vacuoles and deposition of calcium (Farber, 1982;Plaa, 2000). Contributors on liver cell necrosis were deliberated during a symposium," International Meeting On Recent Advances In Biochemical Pathology. Toxic Liver Injury" held at Turin, Italy. These researches were published and edited by Dianzani et al., (1975). Reversal of cell injury using suitable

Apoptosis :

The term apoptosis has been derived from a Greek word meaning "falling off", as the leaves from a tree. It is a synonym to programmed cell death (PCD), a concept proposed by Lockshin (1969). They propounded that cells can commit suicide when required by the organism. It may be an inherent development process or may arise as a response to unknown stimulus. These observations encouraged a search for evidence of gene activation culminating in late 1960s and early 1970s with the recognition that inhibition of protein synthesis can delay or prevent cell death (Lockshin, 1969;Munck, 1971;Martin et al., 1988;Ucker et al., 1989;Openheim et al., 1990). In subsequent years, apoptosis was further classified into intrinsic apoptosis and extrinsic apoptosis.

Intrinsic apoptosis:

It is initiated by a variety of microenvironmental disturbances viz. growth factor withdrawl, DNA damage, endoplasmic reticulum stress, overload of reactive oxygen species, replication stress and micro-tubular defects (Czabotar et al., 2014;Ross et al., 2016;Brumatti et al., 2010). Apoptotic cells retain plasma membrane integrity and metabolic activity (Fig. 2). The critical steps of intrinsic apoptosis are mitochondrial outer membrane permeabilization (MOMP) (Tait and Gram, 2010), which is controlled by proapoptotic and antiapoptotic members of BCL-2 protein family. The inhibitors include BCL-2, BCL-xL, MCL-1, and A1. Whereas, promoters of apoptosis are Bax, Bid and BCL-XS. It is the overall prevalence of BCL-2 or Bax in a cell that will ultimately decide whether a cell will die or be rescued. In physiological conditions, BaX continuously travel between the outer membrane (OMM) and cytosol where it exhibits a quiescent monomeric or inactive dimeric conformation (Edlich et al., 2011;Schellenberg et al., 2013). Intriguingly, BAK resides in the lipid bilayer of OMM. Retro-translocation of BAK from OMM to cytosol has also been reported (Todt et al., 2015). MOMP is antagonized by antiapoptotic members of the BCL2 family. They promote cellular 2+ survival by regulating Ca homeostasis in ER ( Rana, 2020), by promoting bioenergetic metabolism (Bonora et al., 2015) and contributing to the redox homeostasis (Rana, 1997). The interaction between antiapoptotic and proapoptotic BCL2 family members are known to have major therapeutic values (Kotschy et al., 2016).

Extrinsic apoptosis : This signalling pathway involves death receptors viz. TNF family. Members of TNF receptor family share cysteine rich extracellular domains and a cytoplasmic death domain containing about 80 amino acids. Death domain transmits death signal from cell surface to intracellular signalling pathways (Aggarwal et al., 2012). Best characterized ligand and corresponding death receptors include FasL/FasR, TNFα/TNFR1, Apo3L/DR3, Apo2L/DR4 and Apo2L/DR5. The binding of Fas ligand to Fas receptor results in the binding of the adapter protein FADD and TRADD. These events lead to the formation of death inducing signalling complex (DISC) (Dickens et al., 2012). DISC activates procaspase 8. Once caspase 8 is activated, the execution phase of apoptosis is triggered. Caspase 3, caspase 6 and caspase 7 function as executioner caspases. Amongst these, caspase 3 is considered to be most important. Caspase 3 cleaves ICAD to release CAD. CAD degrades chromosomal DNA and causes chromatin condensation.

Autophagy :

The term autophagy or autophagocytosis is derved from a Greek word "autophagos" meaning "self devouring". It is a naturally regulated mechanism that removes unnecessary or dysfunctional components of the cell. Belgian biochemist de Duve (1983) from Louvain University coined this term after his discovery of lysosomes. Eventually he was awarded Nobel Prize in Physiology and Medicine in 1974. It is considered as an adaptive process that protects the cell from nutrient starvation and ATP deficiency. It is manifested through the action of a phagophore, which expands and matures to form an autophagosome. The autophagosome may fuse with endosome and ultimately with a lysosome to form an autolysosome.

It can further be classified as macroautophagy; microautophagy; chaperone mediated autophagy and crinophagy. However, macroautophagy is best characterized primary autophagic pathway. It is primarily a nonselective process, whereas microautography is a selective process. Further, selective autophagy may be termed as mitophagy or peroxypahagy.

S.V.S. Rana: Mechanisms of cell death

It has now been established that in multiple pathological conditions, molecular machinery of autophagy contributes to cell death (Denton et al., 2012;2015;Anding and Baehrecke, 2015). Autophagy dependent cell death may also contribute in the pathogenesis of human disorders. Autophagy promoting factor (APF) has been implicated in the pathogenesis of myocardial infarction owing to its ability to directly promote the expression of ATG7. Another specific variant of autophagy is autosis that relies + + on plasma membrane Na /K ATPase. Inhibition of this enzyme confers neuroprotection in rat model of neonatal hypoxiaischemia (Liu et al., 2013).

Ferroptosis : As the name suggests, ferroptosis is caused by disturbances in the intracellular microenvironment notably lipid peroxidation and iron availability. It occurs independently of caspases, necrosome components of cyclophillin D and autophagy (Dixon, 2017;Yang and Stockwell, 2016;Xie et al., 2016). It exhibits morphological changes viz. mitochondrial shrinkage, reduced or disappeared cristae and ruptured outer mitochondrial membrane (Vanden Berghe et al., 2014).

Ferroptosis inducing agents i.e. erastin (Yang and Stockwell, 2008) and ferroptosis inhibiting agents like ferrostatins and liproxstatins (Hofmans et al., 2016;Friedmann and Angeli, 2014) are known now. Glutathione peroxidase-4 (GPx4) is also considered as the potent endogenous inhibitor of ferroptosis (Seiler et al., 2008). Peroxidation of polyunstaurated fatty acid (PUFA) seems to be regulated by antagonistic behaviour between lipoxygenases (LOXs) which directly catalyze LPO and GPx4 which directly inhibits LPO. Multiple LOXs are involved in LPO and consequent ferroptosis. This cascade of events can be prevented by a few antioxidants viz. ferrostain-1; liproxstatin-1 as well as α tocopherol that function as ROS scavengers. The precise role of this type of cell death in development and cellular/organ homeostasis warrants further studies.

Another group of antioxidant enzymes include glutathione reductase, glucose 6-phosphate dehydrogenase. They are substrate specific and play significant role in cell death.

Non-enzymic antioxidants: Although there are several nonenzymic antioxidants (α tocopherol, ubiquinol-10, β -carotene, bilirubin, ascorbate, glutathione, urate and plasma proteins), best studied amongst these include α-tocopherol, ascorbic acid, glutathione, metallothionein and a hormone melatonin. Vitamin E is a chain breaking antioxidant in biological membranes (Niki , . 1988). It reacts with OH or most commonly with lipid peroxyl . radicals (LOO ) to form a long live α tocopheryl radical. This hypothesis was proved by our experiments made in cadmium and copper fed rat Asorbate was found to be protective against arsenic toxicity ( Now, I would like to introduce the readers of this article to a wonder antioxidant molecule-the metallothionein (MT). This low molecular weight (6.5kD) protein was discovered by Marghose and Vallee (1957) in horse kidney. 30% amino acid residues of MT are cysteines. Therefore, protein has a large number of SH groups that can be coordinated by metal ions. One molecule of MT can bind seven atoms of Cd. I remember classic work done on MT by Cherian (1994) and Klaassen et al. (1999). Protective role of MT against metal toxicity has been studied by our laboratoriy also (Singh and Rana, 2002). However, Rana andKumar, (2000, 2001) confirmed their anti-carcinogenic potential . Recently, protection offered by nanoparticles of zinc against dimethylnitrosamine induced hepatocelluar death was also attributed to MT (Rani et al., 2018).

For many of us, it might be quite surprising to know that a hormone may also play an antioxidative role. Melatonin (N-acetyl-5-methoxytryptamine) is secreted by pineal gland. It functions as a time giver (zeitgeber) in the regulation of circadian rhythms (Arendt, 2003). It synchronizes reproductive cycle in photoperiodic species (Reiter, 1980). An antioxidative protection by melatonin against oxi ime observed that 2+ entry of excess Ca into cardiomyocytes might lead to cardiac pathology and ischemia. Subsequently it was found that receptor overstimulation (Leonard and Salpeter, 1979) or exposure to toxic s (Rana et al., 1997;Rana and Verma, 1997). Singh and Rana, 2010;Sohini and Rana, 2007).

dative injury induced by acetaminophen (Thomas -Zapico and Coto-Mentes, 2005), carbontetrachloride (Ogeturk et al., 2004) and benzene (Sharma andRana, 2010, 2013) has also been reported. Protection offered by melatonin against toxicity induced by several toxic elements was recently reviewed by Rana (2018). Pleiotropic functions of melatonin make it an inevitable molecule.

Pyroptosis :

The term pyroptosis was originally coined by Cookson and Brennan ( 2001) to define a particular type of RCD partially resembling apoptosis but dependent upon caspase 1. It

Role of Cytochrome P : Detoxication of xenobiotics has been

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and still is a subject of prime concern in biochemical toxicology. The parent compound is metabolically converted into a more polar form by phase -I enzymes. Amongst these, the family of CYP enzymes that are found in some prokaryotes and all 450 eukaryotes play a paramount role in oxidative, reductive and peroxidative biotransformation of many endogenous and exogenous compounds. A German biochemist Klingenberg (1958) species (Shimamoto, 2013). Significance of reactive oxygen species (ROS) is fundamental to our understanding on cell death.

Reactive oxygen species and cell death : Reactive oxygen species (ROS) namely, the superoxide anion, hydrogen peroxide, singlet oxygen, nitric oxide, hydroxyl radicals are the products of normal aerobic metabolism. Biochemical processes like oxidation/peroxidation, inflammation, phagocytosis, exposure to ultraviolet radiation and xenobiotics lead to generation of ROS. Fenton or Haber Weiss reactions too, contribute to increased formation of ROS. They can cause oxidative DNA damage, protein oxidation and lipid peroxidation (LPO). LPO, today is central to our understanding on cell injury. It is by and large accepted that membrane damage by xenobiotics is the gateway for the entry of xenobiotics in the cell. Landmark paper from Recknagel and Ghoshal (1966) attributed cell injury to peroxidative damage of polyunsaturated fatty acids (PUFA) caused by xenobiotics. Subsequent papers from his group unfolded the mystery of carbon tetrachloride induced liver inury (Recknagel, 1967;Recknagel et al., 1982). Tappel (1973) defined this phenomenon as oxidative deterioration of PUFA. Free radical mediated peroxidation of PUFA can take place through different mechanisms. This mechanism of cell injury was considered central to the pathogenesis of disease (Farber, 1982). The role of free radicals in biology and medicine was very precisely discussed by Halliwell and Gutteridge (1985) in a popular book, "Free Radicals in Biology and Medicine". This book revolutionized the concept of cell death.

Lipid peroxidation and consequent events induced by heavy metals and organic solvents mainly in the liver and kidney

O n l i n e C o p y 910

Role of calcium in cell death :

We know that calcium is an essential element. It governs a host of vital cell functions and hence is necessary for cell survival. Intriguingly, it has also been 2+ established that cellular Ca overload may contribute in cell death. Fleckenstein et al. (1974) for the first t 2+ agents (Trump and Berezesky,1995) (Wyllie, 2+ 1980). Major effects of increased Ca are activation of calpains, endonucleases and calcium dependent phospholipases, in particular phospholpiase A2 that lead to necrotic cell death. Rana and Rastogi (1991) showed that parathyroidectomy offered protection against carbon tetrachloride induced hepatotoxicity through inhibition of phospholipase A2. A number of papers on 2+ role of Ca in apoptosis has been published (Orrenius et al., 2003;Orrenius and Zhivotovsky, 2006;Orrenius et al., 2007;Orrenius et al., 2011 andOrrenius, 2019). The link between 2+ apoptosis and Ca during heavy metal toxicity was reviewed by Rana (2008). More recently, ER stress induced by heavy metals has also been linked to calcium dependent processes (Rana, 2020).

Cell death and redox cycle : Reductive stress (RS) is the counterpart of oxidative stress (OS). It can occur as a response to conditions that shift the redox balance of important biological (Jones and Sies, 2015). The redox code is a critical complement of the genetic code, the epigenetic code and the histone code in the molecular logic of life (Oronsky et al., 2014). Redox code is manifested by redox switches and redox active metabolites such as H O , nitric oxide or hydrogen sufide. These 2 2

molecules are now known to play a pivotal role in cellular death. Impressive research on these molecules have been performed by Prof Helmut Sies from University of Dusseldorf, Germany ( Sies and Summer, 1975;Sies, 1986;Sies and Jones, 2007) , Prof Hideo Kimura from Sanyo Onoda City University, Yamaguch, Japan ( Kimura 2013( Kimura , 2015) ) and a Nobel awardee Prof Gregg L Semenza from Johns Hopkin University School of Medicine ( Semenza, 2005( Semenza, , 2007)). (Anderson et al., 1981). Several chemicals can cause mutations in mTDNA (Valente et al., 2016). In turn, these mutations increase the sensitivity of mT to stress (Chan, 2017;Luz et al., 2017).

Stress can change the morphology and function of mT through fusion and fission (Meyer et al., 2017). Fig 3 exhibits these morphological changes. Role of reactive oxygen species in mT mediated cell death was reviewed by Orrenius (2007). For further information, readers are advised to consult reviews written by Meyer et al., (2013); Meyer and Chan, (2017); Dykens and Will (2007).

Endoplasmic reticulum (ER) stress and cell death : Several drugs/chemicals are now known to induce ER stress. It could lead to deleterious effects within cells and tissues including accumulation of lipids, cytolysis, inflammation, energy depletion, redox imbalance and cell death (Malhi and Kaufman, 2011;Coop et al., 2012;Cheng et al., 2013;Chen et al., 2014). Since many xenobiotics are reactive, their influence on structure and function of ER is inevitable. ER stress is expressed by accumulation of unfolded or misfolded proteins in the luman of ER (Byrd and Brewer, 2012). ER stress is known to contribute in the pathogenesis of several diseases viz. diabetes, neurodegenerative disorders, inflammatory and autoimmune diseases (Hetz et al., 2013;Wang and Kaufman, 2012).

A mechanism that modulates ER function is known as unfolded protein response (UPR). ER stress and generation of ROS are fundamental to UPR signalling. ROS can trigger UPR by themselves (Santos et al., 2009). ER stress plays an important role in the toxicity of heavy metals (Rana, 2020). ER maintains a close contact with mitochondria (Fig 4). This contact facilitates regulated transfer of calcium between the two organelles. It is assumed that too much or too little cross talk between two might contribute to metabolic dysfunction (Patergnani et al., 2011).

Global gene expression profiling :

Toxicology underwent a major technological transformation in its ability to generate global gene expression profiling data on a massive scale. High throughput techniques viz. toxicogenomics, proteomics and metabolonomics ushered a new era in molecular toxicology (Hamadeh and Afshar, 2004). Genes and proteins responsible for cell death caused by a variety of xenobiotics have now been identified (Jeong et al., 2006;Lim et al., 2007;Park et al., 2008;Heo et al., 2010). Molecular markers of cell death are being identified. Two reviews on these efforts were written by me along with colleagues of Korea Institute of Toxicology, Daejeon (South Korea) where I worked in 2006 as a fellow of Korean Education Foundation (Yoon et al., 2007;Yoon et al., 2008). The concept of "Molecular markers in Health and Disease", was the focus of a plenary lecture delivered by me during International Conference of Toxicogenomics and Proteomics," held at Incheon, South Korea from 1-2 November 2007 (Rana et al., 2007). Tox-21 (Toxicity Testing in 21st Century), initiative taken by NRC and it collaborators aims to advance the molecular understanding and predictability of toxic responses that would help in safeguarding human health from a variety of poisons.

Cross talk amongst different mechanisms of cell death:

Above account signifies that multiple signal transduction cascades are responsible for RCD of a variety of cell types. Majority of drugs/chemicals/xenobiotics are capable of producing ROS. In addition they induce oxido-reductive stress, mitochondrial stress and ER stress. The cross talk amongst these mechanisms has recently been debated in a few reports. The interorganelle cross talk apparently involves several "molecular switches" within the signalling network. Furthermore, nature and severity of the stimulus, cell type and hierarchy of interorganelle cross talk might result in different cell death modalities. For example, depletion of energy metabolism may change the mode of cell death from apoptosis to necrosis. Similarly, inhibition of caspase enzyme might change apoptosis to necrosis or autophagy (Yousefi et al., 2006;Orrenius et al., 2011). ER stress can also cause apoptosis as well as autophagic cell death (Orrenius et al., 2003). Intracellular accumulation of calcium can trigger necrotic cell death but surviving cells may subsequently succumb to apoptosis. Taken together, it is important to delineate a specific mechanism involved in the toxicity of a specific xenobiotic. Intriguingly, all these mechanisms are interdependent and work as closely knitted network of signalling pathways as seen in necroptosis. In a few instances, cell death mechanisms share common signals. Dev. Cell., 38, 536-547 (2016). Kurz, T., A. Terman, B. Gustaffson and U.T. Brunk: Lysosomes and oxidative stress in ageing and apoptosis. Biochim. Biophys. Acta., 1780, 1291-1303(2008). Leonard, J.P. and M.M. Salpeter: Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J. Cell Biol., 82, 811-819(1979). Linossier, G.: Contribution a l'etude des ferments oxidants Sur la peroxidase du pups. C. R. Soc. Biol. Paris., 50, 373-375(1898) Arh Hig Rada Toksikol., 51, 279-286 (2000).