Research Progress of Programmed Cell Death Induced by Acrylamide

Acrylamide exposure through environment pollution and diet is very common in daily life. With the deepening of the study on the toxicity of acrylamide, it has attracted widespread attention for the efects of acrylamide on multiple organs through afecting a variety of programmed cell death. Multiple studies have shown that acrylamide could exert its toxic efect by inducing programmed cell death, but its specifc molecular mechanism is still unclear. In this review, the research on the main forms of programmed cell death (apoptosis, autophagy, and programmed necrosis) induced by acrylamide and their possible mechanisms are reviewed. Tis review may provide basic data for further research of acrylamide and prevention of its toxicity.


Introduction
Food safety involves not only the toxic and harmful substances added to food in various food processes but also a series of harmful substances produced by some chemical reactions in food production.Acrylamide (AA) has been detected in potato chips, cofee, and some grain foods, such as all kinds of cakes, cookies, bread, and toast (Figure 1) [1,2].Te content of AA in diferent foods is listed in Table 1.Te content of AA in potato crisps, bread, cofee, and biscuits is very high.Moreover, the content of AA in these fried or baked foods is much higher than the content standard (0.5 μg/L) in drinking water stipulated by the World Health Organization.Table 1 shows that the level of AA in diferent food changed depending on the ingredients and processing conditions.Several pathways of AA formation in food have been reported [3,4], but the most probable pathway of AA formation in food is the Maillard route (Figure 2).Asparagine is considered to be the most important precursor of AA [5,6].When asparagine is heated alone, a very limited amount of AA is formed.So it needs a carbonyl group to accelerate its conversion to AA. Te classical pathway (Figure 2(a)) occurs in the heating of asparagine in the presence of compounds that have α-hydroxyl carbonyl groups (such as the reducing sugars), which leads to the formation of Schif bases in the Maillard reaction.Schif bases can be decarboxylated directly by Schif betaine or indirectly by an intermediate of oxazolidin-5-1 to form azomethine ylide.Afterward, AA may be produced directly from azomethine ylide, through the deamination of 3-aminopropionamide (3-APA), which is regarded to be a direct precursor of AA.In addition to compounds containing carbonyl α-OH groups, other active carbon groups may also take a part in the formation of AA [7].When α-dicarbonyl groups are available, alternative pathways (Figure 2(b)) appear to be activated.A signifcantly related reaction is the Strecker degradation of amino acids by these intermediates, in which an aldehyde is formed through decarboxylated and deaminated of amino acid.Terefore, food exposure is a common way.In addition, chemical poly-AA synthesized from AA are widely used in the treatment of drinking water and industrial wastewater, oil mining, papermaking, production of textile, adhesive, dye and cosmetics, and other felds.In the process of polymer synthesis and application, a small amount of AA monomer may inevitably remain or decompose, polluting the atmosphere, water, and soil [8][9][10][11][12][13].
A tide of research shows that AA can be absorbed through the digestive and respiratory tract, skin, and mucous membranes and transported to various body tissues through the blood.AA even enters into fetuses and infants through the placenta and milk [14,15].AA has been reported to produce reproductive toxicity, developmental toxicity, neurotoxicity, immunotoxicity, and carcinogenicity after entering the human and animal body [14,[16][17][18][19]. Terefore, the research and control of the toxicity of AA have had a high profle around the world.
Te toxicity of AA not only has a broad spectrum but also causes severe harm.Te research regarding to the toxic AA is no longer simply to evaluate its harmful efects on organisms, but also to explore the specifc toxic mechanism.A plethora of studies have already suggested that one of the critical mechanisms of AA is to trigger programmed cell death (PCD).PCD refers to an orderly and active way of cell death to maintain homeostasis under the stimulation of certain signals or factors.Apoptosis, autophagy, pyroptosis, and necroptosis are common ways of PCD [20].AA was previously found to induce cell apoptosis.Recent studies have shown that AA could still lead to cell autophagy.Tere is also evidence that AA-induced injury may be related to cell pyroptosis and necroptosis.Tis review mainly summarized the research of AA-induced PCD and may provide clues for the elucidation of the possible toxic mechanism and the risk control of AA.

Acrylamide Induces Cell Apoptosis
Apoptosis is an early, chronic, and mild reaction after injury induction.It is a highly regulated and gene PCD process during cell growth and development.It maintains the  2 Journal of Food Quality homeostasis of cells and biological organism by clearing of redundant, aging, and damaged cells [21,22].Te classic apoptotic pathway includes three major signaling pathways: the extrinsic death receptor-induced pathway, the intrinsic mitochondria-mediated pathway, and endoplasmic reticulum stress pathway [23].Apoptosis is an active process in the body, which is regulated by apoptosis-related genes.
During the process of apoptosis, the bcl-2 family proteins located in the mitochondrial membrane were changed, including the expression level of proapoptotic protein bax increased and the expression level of antiapoptotic protein bcl-2 decreased.It has been found that AA can induce apoptosis in nerve cells, germ cells, retinal cells, hepatocytes, bone marrow cells, and so on, endangering the growth and function of cells.

Acrylamide Induces Apoptosis in Nervous Related Cells.
An increasing number of evidence proved that AA caused severe neurotoxicity.Although the neurotoxic mechanism of AA is complex, the induction of neuronal apoptosis represents as one important mechanism.Te research of apoptosis has no longer only focus on nerve cells, and more research has gone deep into the tissues and cells related to the nervous system.Te apoptotic mechanism has been deeply explored, which provides an important scientifc basis for intervention and prevention of AA-induced injury.Previous animal studies have suggested that there was an early apoptosis detected in AA-induced neuropathy.For example, the results of a subchronic model in Wistar rats and SD rats showed that after exposure to AA, the expression of antiapoptotic bcl-2 decreased in the cerebral cortex and cerebellar cortex, while the expression of proapoptotic bax increased and caspase-3 was activated [24][25][26].Typical apoptotic characteristics such as nuclear pyknosis occurred in cerebral cortical neurons.Tere were diferent degrees of the apoptotic phenomenon in the central and peripheral nervous system.Meanwhile, the expressions of bax and caspase-7 in cerebellum of rats exposed to AA (5 mg/kg) signifcantly increased, and apoptotic phenomenon were observed in more than half of the isolated cells [27].
Some researchers have suggested that AA-induced apoptosis occurs in the Purkinje cell layer and the cerebellar cortical granulosa cell layer, due to the observed condensation and pyknosis of the nucleus [28][29][30][31].Purkinje cells are the largest neurons in the cerebellar cortex and the only neurons that can transmit impulses from the cerebellar cortex.Te nuclear factor erythroid 2-related factor 2 (Nrf2) family is a transcription factor that regulates the redox state of cells and is involved in the coordination of adaptive responses to various stimuli [32].It has been found that AAinduced degeneration and apoptosis in cerebellar Purkinje cells were related to the inhibition of the Nrf2 pathway [33].
It has also been reported that the occurrence of nerve cell apoptosis is mediated by the PERK pathway activated by ERS (endoplasmic reticulum stress) [28].Zebrafsh is an ideal model to study the development, function, and tissue repair of the nervous system.AA also exerted severely toxic efects on zebrafsh.Zebrafsh after being exposed to AA experienced an injury in its brain structure, and this injury was due to cell apoptosis mediated through ERS and eIF2α-ATF4-CHOP signal cascade [34].In SH-SY5Y cells, AAinduced phosphorylated tau protein aggregation, phosphorylated cAMP response element binding protein (CREB) decreased, and bax/bcl-2 ratio was up-regulated.AA could also activate PERK-eIF2α pathway in SH-SY5Y cells, trigger the activation of glycogen synthase kinase-3β (GSK-3β), and up-regulate activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) [35].
Te central nervous system has active oxygen metabolism, but the activity of antioxidant enzymes is relatively low.Terefore, oxidative stress plays an important role in AA-induced neurotoxicity.Numerous studies have shown that AA can induce neuronal apoptosis in various neural cell models through mitochondrial and death receptor pathways [36][37][38].For example, AA could down-regulate the expressions of miR-21, p-Akt, and bcl-2 in SH-SY5Y cells and up-regulate the levels of PTEN, bax, caspase-9, and caspase-3, thus triggering mitochondrial oxidative stress [39].Mitogen-activated protein kinase (MAPK), a serinethreonine protein kinase including extracellular regulated protein kinases (ERK), c-JunN-terminal kinase (JNK), and p38 protein, could control apoptosis and proliferation of cell [40,41].Te mitochondrial apoptosis induced by AA in PC12 cells was closely related to the activation of JNK and p38 pathways [37].Nuclear factor-κB (NF-κB) could regulate a variety of target genes including those associated with proliferation and apoptosis.Moreover, the NF-κB signal is prone to crosstalk and afects a variety of signaling pathways [42].AA could also cause the accumulation of cellular reactive oxygen species (ROS) by increasing the mRNA level of ERS-dependent apoptosis factor C/EBP homologous protein (CHOP) and inactivating the NF-κB pathway [34,43].Research has proved that AA could activate the NF-κB cascade, increase the ratio of Bax/ Bcl-2, and cause the cleavage of caspase-3, caspase-9, and PARP to induce apoptosis [44].
In addition, a whole cell model in vitro was established to simulate the barrier and metabolic microenvironment in the nervous system in vivo.A noncontact cocultured bloodbrain barrier (BBB) model in vitro was established with human umbilical vein endothelial cells (HUVEC) and rat glioma cells (C6).Human renal cortical proximal convoluted tubular epithelial cells (HK-2), human normal hepatocytes (L-02), and human neuroblastoma cells (SH-SY5Y) were inoculated into a cell coculture plate to establish an integrated discrete multiple organ cell coculture (IdMOC) model.Ten, SH-SY5Y cells were either directly exposed to AA, or indirectly exposed to AA via the BBB model and IdMOC model.Te results showed that the SH-SY5Y apoptosis rate of the indirect exposure group was signifcantly lower than that of the direct exposure group [45].
Tere are also a large number of neuroglia in the nervous system, such as astrocytes, oligodendrocytes, microglia in the central nervous system, and Schwann cells in the peripheral nervous system.Astrocytes play the role of supporting and separating nerve cells and participate in the formation of the blood-brain barrier.Te toxic efects of AA on rat primary astrocytes and three human astrocytoma cell lines (U-1240MG, U-87MG, and U-251MG) have been tested.It was found that, after exposure to 2 mmol/L of AA for 48 h, an increased ratio of bax/bcl-2 was detected in primary astrocytes and U-87MG cells, whereas an overexpression of bcl-2 was observed in U-1240MG and U-251MG cells.Te levels of p53 and p-p53 in primary astrocytes increased, and caspases (caspase-3, caspase-8, and caspase-9) were activated in all cell types.Tese results indicate the existence of a common apoptotic pathway among all astrocytes, and U-87MG cells might be a suitable in vitro model besides primary astrocytes [46][47][48].In response to various cellular stressors, such as oxidative stress, hypoxia, DNA damage, RNA consumption, and oncogene activation, the tumor suppressor gene p53 may be activated and overexpressed, promoting cell apoptosis [49].Te latest research shows the increased apoptosis induced by AA may be due to the high increase of the p53 expression level [50].
In addition, the results in mice found that the apoptotic efect of AA on astrocytes was closely related to the deletion of metallothionein I/II (MTI/II) [51].AA could also impair the energy metabolism of mouse microglia cell line BV2 by reducing mitochondrial respiration, anaerobic glycolysis, and the expressions of complex I, III, and IV subunits, leading to apoptosis [52].Recent research using the zebrafsh model also reported that AA could enhance microgliainduced neuronal apoptosis by impairing the capacity of oxidative repairing systems [53].
AA could also induce apoptosis in other nerve cells.For example, AA could induce apoptosis in adult hippocampal neurons and pluripotent neural precursor cells in mice [54].AA in HT22 (mouse hippocampal neuron cell line) cells could down-regulate the bcl-2 level and up-regulate the levels of bax and cleaved caspase-3 [55].AA could also stimulate apoptosis in motor neuron VSC4.1 cells (Spinal cord anterior horn motoneuronoma cell line) by elevating the expressions of GRP78 (glucose-regulated protein 78), ATF6 (activating transcription factor 6), and IRE1 (Inositolrequiring enzyme 1), which indicated the role of endoplasmic reticulum stress in regulating apoptosis [56].Recently, cerebral organoids based on human embryonic stem cells (hESC) have been used to analyze human neurodevelopmental toxicity.Te results by hESC-derived cerebral organoids showed that AA could signifcantly interfere with the transcriptional profle, elevate Nrf2-mediated gene expression, cause cell apoptosis, and promote tau hyperphosphorylation in cerebral organoids [57].[58][59][60].AA could also disrupt the dynamic balance of spermatogenic cell division, proliferation, and apoptosis in male rats and then cause spermatogenesis disorder.Te specifc mechanism is closely related to the reduced protein expressions of XRCC1, TERT, and PCNA, as well as the declined activity of the antioxidant enzyme (SOD, CAT, and GPX) in testicular spermatogenic cells, and the mitochondrial damage in testicular seminiferous tubules [61][62][63].Recent studies suggested that AA-promoted apoptosis in rat testicular tissue was also mediated by stimulating p38α-MAPK, TNFα, and PI3K/Akt/mTOR pathways [64].Notably, AA exposure could also induce DNA damage and oxidative stress in mouse sperm.Tus, these may be one of the reasons why H2AX phosphorylated amplifcation and meiosis processes are interrupted.It is illustrated that MVHpositive cells decreased in AA-treated mice.In conclusion, DNA damage and oxidative stress may be one of the causes of germ cell apoptosis and further lead to germ cell depletion [65].
Leydig cell is an endocrine cell located in the mammalian testicular stroma.It plays an important role in promoting the diferentiation and development of embryonic reproductive organs, as well as maintaining sexual function and promoting metabolism.It is already found that AA could destroy antioxidant systems and induce the mitochondrial apoptosis pathway in Leydig cells, resulting in sperm defects and various abnormal histopathological injuries [66,67].Recent fndings suggest that AA exposure could lead to structural and functional damage of Leydig cells and mouse testis, and decreased testosterone synthesis, which may be related to activation of ERK1/2 phosphorylation [68].
AA can also damage female fertility.Prenatal development is extremely sensitive, and the efect of any poison may permanently damage its process.AA could easily pass through the placenta and breast milk.It has been reported that maternal exposure to AA could lead to weight loss at birth, and the decrease of follicles as well as oocytes apoptosis in the ofspring of newborn guinea pigs [69,70].AAcaused follicular cell apoptosis was responsible for ovarian dysfunction [71].Evidence by evaluating the efect of AA on apoptosis-related genes in the ovaries showed that the most increased ratio of Bax/Bcl-2 was found in the AA group compared to the normal group.AA may induce ovarian dysfunction by increasing the proportions of apoptosisrelated genes [72].It has been reported that AA exposure may inhibit the endocrine function of lutein in pregnancy through ovarian oxidative stress and apoptosis [73].AA could also promote the apoptosis of mouse extracellular cumulus granulosa cells, delay cell maturation, and decrease developmental potential [74].Some results are indicated that AA exposure reduces the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin assembly abnormalities, and apoptosis, which impair chromatin structure, sperm binding capacity, and embryonic development [75].
Decidualization is an important process of a successful pregnancy.It is reported that AA could signifcantly inhibit the decidualization of mouse endometrium by inducing apoptosis [76].AA during pregnancy mainly caused placental development arrest by regulating the expressions of key placental AA genes and labyrinthine vessels, inhibiting proliferation and inducing apoptosis.Moreover, P-ERK1/2 and p53 may be involved in the process [73,77].In addition, it is also shown that benzopyrene had an obvious synergistic efect on gonadal cell apoptosis in Caenorhabditis elegans [78].

Acrylamide Induces Cell Apoptosis in Bone Marrow/Spinal
Cord Tissues.Bone marrow mesenchymal stem cells (BMMSCs) participate in a variety of immune responses and have the ability to preferentially migrate to repair damaged tissues and organs in vivo.Someone reported that after AA treatment of BMMSCs for 72 h, the levels of ROS, HSP27, and IL-8 increased signifcantly, and NF-κB pathway was activated, but the cell cycle and apoptosis hardly changed [79].Others found that the structure of spinal cord tissue was destroyed and obvious characteristics of apoptosis appeared after AA exposure.BMMSCs transplantation can inhibit the spinal cord cell apoptosis caused by AA, and its mechanism may be related to promoting the expression of bcl-2 and inhibiting the expression of bax [80].It is still shown that AA can damage bone development and remodeling, which was due to the apoptosis of mesenchymal progenitor cells (HMPC) [81].

Acrylamide Induces Apoptosis in Gastrointestinal Cells.
AA is also toxic to the gastrointestinal tract.Male mice (BALB/c) exposed to AA displayed alteration of morphology and histology of the small intestinal wall and decrease in proliferation, villus length, fractal dimension, crypt depth, and number, as well as the small intestinal absorptive surface.Conversely, there was an increase in apoptosis and parameters associated with nerve ganglia [82].

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It has been reported that AA caused mucosal erosions and depletion of the protective surface mucus as well as widespread infammatory infltration in adult male albino rats.A significant increase in the expressions of caspase-3 and iNOS indicated the involvement of apoptosis and oxidative stress.Rosemary extracts exerted a protective efect against AAinduced gastric apoptosis and infammation [86].

Acrylamide Induces Apoptosis in Liver, Kidney and Lung
Cells.It has been previously reported that AA caused apoptosis in HL7702 cells (human hepatocyte) by the disturbing cell cycle, disrupting DNA function and activating protooncogene c-jun and c-fos [87].Studies have shown that AA induces hepatocyte apoptosis, leading to an increase of bax, TGF-β1, and COX-2 and decreases the expression of bcl-2, Nrf2, HO-1, and antioxidant [88].AA also stimulated apoptosis in HepG2 cell, rat liver cells (e.g., BRL-3A cells and IAR20 cells), and kidney cells, and the miR-27a-5p-Btf3-ATM-p53 axis might play a vital role in the promotion of AA-induced cell apoptosis through disrupting mitochondrial structure and function [89].It was reported that the application of AA increased the level of KIM-1 in kidney tissue, as well as the expression levels of caspase-3 and bcl-2.In this study, it was observed increased expression levels of NF-κB and MAPK-1 in the renal tissue of rats treated with AA, suggesting that AA can trigger infammation and apoptosis of renal tissue by stimulating NF-κB and MAPK-1 [90].Meanwhile, the apoptosis could be inhibited by silymarin, morin, and rosmarinic acid by regulating the key proteins of the IRE1 pathway (p-IRE1α, XBP-1s, and TRAF2), PI3K/Akt/mTOR signal pathway and the expression of endoplasmic reticulum stress (ERS) characteristic proteins (GRP78, p-ASK1, Caspase-12, and CHOP) [91,92].
Te respiratory system was also reported to be damaged by AA.BEAS-2B cells (pulmonary epithelial cell) after AA treatment displayed obvious apoptotic characteristics, such as the signifcantly decreased ratio of bcl-2/bax, and the increased level of Nrf2 expression and caspase-3/7 activity [93].It is also found that the mRNA expression levels of proapoptotic Bax and procaspase-3 signifcantly were higher after AA exposure, and the levels of antiapoptotic Bcl-2 were relatively lower.Moreover, the ratio of either p-ERK/ERK or p-JNK/JNK was signifcantly elevated by AA [94].

Acrylamide Induces Cell Apoptosis in Immune System.
AA has been discovered to threaten the immune system.AA had adverse efects on peripheral blood lymphocytes (PBL) and intestinal associated lymphoid tissue (GALT) in male SD rats [95].AA can activate caspase-3 in human lymphocytes and human monocyte macrophages and cause PARP fragmentation, inducing apoptosis [96,97].
Te results in the mouse model showed that AA could reduce the weight of thymus and spleen and cause pathological atrophy, the imbalance of the proportion of peripheral blood lymphocyte subsets, and the decrease of cytokine levels [98].Meanwhile, the apoptosis rates of the spleen and thymus in the middle and high-dose groups of AA were signifcantly higher than those in the normal group.
AA-induced a large number of apoptosis in mouse splenocytes through the cascade activation of caspases.Because AA caused a disorder of mitochondrial electron transfer chain complexes I and III, accompanied by the collapse of mitochondrial membrane potential and the accumulation of ROS [99].

Acrylamide Induces Apoptosis in Cell Associated with
Visual Disorder.According to statistics, professional workers and experimental animals exposed to AA endure visual impairment, but the mechanism has not been clarifed.Studies have confrmed that AA could induce slight or severe apoptosis in bovine lens epithelial (BEL) cells, human retinal pigment epithelium (RPE) cells, and the rod cells and cone cells of zebrafsh embryos.Tese processes are caspase-3-dependent, accompanied by increasing ROS, inactivating antioxidant enzymes, and inhibiting Gpx1 and Nrf2 [100][101][102].

Acrylamide Induces Cell Apoptosis in Other Systems.
AA could also afect preneoplatic lesions of the urinary tract in mice.Abnormal apoptotic/mitosis ratio and the expression of caspase-3 increased in AA-treated mice, and dietary PUFA (polyunsaturated fatty acid) such as n-6 PUFA (corn oil) could modulate preneoplastic proliferation in AAtreated mice [103].Replicative senescence, characterized by a limited ability of cells to divide in vitro, induces endothelial dysfunction.Many compounds in food can induce earlier vascular senescence.Chronic exposure to lower concentrations of AA induces accelerated senescence and causes endothelial dysfunction in vivo.Since AA in vitro could inhibit HUVEC proliferation and induce apoptosis in doseand time-dependent manners [104].
Apoptosis occurs continuously during tissue development to remove abnormal cells while causing minimal damage to surrounding tissues.Apoptosis can be upregulated by diferent signal transduction pathways under the induction of AA.Te death receptor pathway, mitochondrial pathway, and ER stress pathway are all involved (Figure 3).Te search for a new target as a key regulator of apoptosis signals may provide further insights into the intervention of AA toxicity.

Acrylamide Disturbs Cell Autophagy
Autophagy refers to the process in which some damaged proteins or organelles are encapsulated by autophagy vesicles with double-layer membrane structure and then sent to lysosomes (animals) for degradation and recycling [105].Autophagy-related proteins include LC3 and Beclin-1.LC3-I and LC3-II are two forms of LC3.LC3-II is transformed from LC3-I by binding with phosphatidyl ethanolamine, which is a marker of autophagosome, and Beclin-1 is a key protein regulating the formation of autophagosomes.Specifcally, autophagy fow is a fnely regulated process, including the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of autophagolysosomes.AA can induce varying degrees of autophagy in diferent cells.

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Acrylamide Induces Cell Autophagy in Nervous Systems.
In the study of AA neurotoxicity, a large number of autophagosome, swollen mitochondria, and enlarged endoplasmic reticulum could be observed in the cerebellum of AA-exposed rats.Te ratio of LC3-II/LC3-I and the protein expression of Beclin1 were signifcantly elevated in AA-exposed rats.ATF4 and CHOP, as well as PERK-mediated endoplasmic reticulum stress (ERS), participated in AA-induced autophagy [28].It has also been reported that AA regulates the autophagy signaling pathway in U87-MG cells by increasing the expression of p62 and Beclin-1 and decreasing the protein expression ratio of LC3-I/LC3-II [44].In vitro study with PC12 cell, it was found that AA promoted the protein expressions of Beclin-1, LC3-II as well as p62.AA-induced neuronal death was weakened by an autophagy inhibitor (3-methyladenine) and worsened by a lysosomal inhibitor (chloroquine), proving that AA could severely disturb the autophagy homeostasis.In addition, it was also found that AA-induced autophagy by activating CYP2E1, ERK, PKC-α, and PKC-δ and inhibiting AMPK, p38, and JNK pathways.Pretreatment with bioactive polyphenols blocked AA-stimulated LC3 transformation and autophagy activation [19,106].

Acrylamide Induces Cell Autophagy in Reproductive
Systems.Te toxic mechanism of AA in reproductive and developmental systems still involves cell autophagy.Prenatal AA exposure signifcantly caused the reduction of the number of primordial follicles and primary follicles in neonatal guinea pigs.Tere were not only apoptosis signals but also slight autophagy signals observed in oocytes of primary and secondary follicles [69].After rats (female Wistar-Albino of ffty days old) were exposed to AA (2.5, 10, and 50 mg/kg/day), ovarian weight and the concentrations of serum progesterone and estradiol signifcantly decreased.A high dose of AA (50 mg/kg/day) signifcantly induced the overexpressions of INSL3, CYP17α, IGF1, ESR1, ESR2, ATG5, ATG12, and LC3 in the ovary.In addition, AA-induced ovarian dysfunction was mediated by infuencing steroid hormone release and activating mRNA levels of autophagy-related genes [71].AA could also down-regulatebcl-2 and up-regulate protein levels of p38-α MAPK, TNF-α, NF-κB, IL-1β, IL-6, COX-2, cytochrome c, bax, Caspase-3, LC3, and Beclin-1 in testicular tissue/cell of male rats, indicating a large number of apoptosis and autophagy.Morin can interfere with AA-induced apoptosis and autophagy by regulating PI3K/Akt/mTOR and NF-κB signal pathway [64].Furthermore, autophagy occurred in the oocytes of mice exposed to AA. Tese results suggested that AA exposure led to the reduced developmental potential of mouse germinal vesicles (GV) by damaging chromatin structure, sperm-binding capacity, and embryonic development through autophagy [75].

Acrylamide Induces Cell Autophagy in Liver and Kidney
Injury.AA-induced hepatotoxicity and nephrotoxicity in rats is also related to excessive autophagy.Not only the obvious characteristics of apoptosis but also the autophagy indexes such as Beclin-1 and LC3 were signifcantly upregulated in the liver and kidney of AA-treated rats.In addition, Morin also reversed the changes in levels of apoptotic and autophagic parameters by regulating p38-α MAPK, NF-κB, and PI3K/Akt/mTOR pathway [91].

Acrylamide Suppresses Cell Autophagy.
Besides induction of excessive autophagy, AA also inhibits normal autophagy.For example, in AA-treated U2OS cells (human  osteosarcoma cells), autophagy was inhibited although the apoptosis rate increased.Song et al. reported that AAinduced the accumulation of autophagy markers LC3-II and p62, suggesting that AA may inhibit autophagy.It seems that the toxic mechanism of AA-induced autophagy is complex and cell-specifc [107].
Apoptosis and autophagy have been observed simultaneously in AA-induced cytotoxicity.Generally speaking, AA stimulates cell apoptosis and autophagy in the liver, kidney, and nerve as well as germ cells, AA-stimulated cell apoptosis, and autophagy can be mediated by some common signal pathways.For example, AA-induced autophagic accumulation may be attributed to the blocking of autophagic fux, preventing the autophagic from binding to the lysosome.Additional information confrming the association between apoptosis and autophagy fux suggests that limiting the protective autophagy induced by AA further promotes apoptosis initiation [44].However, the level of autophagy changes greatly sometimes, and the relationship between apoptosis and autophagy during the AA-induced damage process has not been fully elucidated.
Autophagy is an important cellular mechanism involved in a variety of cellular processes.Autophagy helps maintain cellular homeostasis under appropriate stress conditions.Either autophagy inhibition or promotion will afect the normal proliferation and function of cells.It may also induce apoptosis when stress is prolonged or aggravated.In summary, AA-induced autophagosome accumulation may be due to the blocking of autophagic fux, which prevents the binding of autophagosomes to lysosomes, and excessive autophagy may also activate apoptosis (Figure 4).

Acrylamide Causes Cell Pyroptosis
Cell pyroptosis has attracted more and more attention in recent years.Te morphological characteristics, occurrence, and regulation mechanism of cell pyroptosis are diferent from other types of cell death [20,108].Pyroptosis is a kind of caspase-1-dependent and proinfammatory form of cell death, accompanied by the release of a large number of proinfammatory factors.Pyroptosis appears morphologically to be a combination of apoptosis and necrosis, involving the destruction of plasma membrane integrity and the release of cytoplasmic contents.During cell pyroptosis, the size of dying cells increases signifcantly and the nucleus becomes round and shrinks with the expansion of cells.Like apoptotic cells, the DNA fragments of pyroptotic cells were positive under TUNEL staining.Unlike apoptotic cells, pyroptosis cells maintained nuclear integrity.

Acrylamide Induces Cell Pyroptosis in Nervous Systems.
In the study of AA-inducedneuro-degeneration in mice (20 mg/kg body weight for 4 weeks), it was found that proinfammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) increased, indicating the possible existence of pyroptotic events [109].NLRP3 (Nucleotide-binding oligomerization domain, leucine rich repeat, and pyrin domain containing 3) infammasome, an intracellular multiple protein complex, is composed of the sensor protein NLRP3, the adaptor apoptosis-associated specklike protein (ASC) and the cysteine protease-1 precursor (procaspase-1).NLRP3 infammasome when assembling forms the effector protein caspase-1.Ten, caspase-1, as a precursor of infammatory caspase, cleaves the cytokines such as pro-IL-1β and pro-IL-18 into pro-infammatory cytokine IL-1β and IL-18.Tus, NLRP3 plays an important role in caspase-1-dependent pyroptosis pathway.AA exposure stimulates the activation of NLRP3 infammasome mainly through MARK, Nrf2, and NF-κB pathways [110].Te activation of NLRP3 infammasome and its subsequent downstream infammatory response have been detected in AA-induced neurotoxicity.For example, in AAinduced BV2 cell death, there was an obvious activation of NLRP3 infammasome and an increase of cytokine interleukin-1β and interleukin-18 expression.Tese were also observed in AA-exposed C57BL/6 mice and SD rats.Either intervention with specifc NLRP3 inhibitor MCC950 or NLRP3 knockout signifcantly reversed AAinduced cerebellar Purkinje cell degeneration and pyroptosis.AA-induced pyroptosis was also accompanied by NF-κB activation, and a signifcant increase of infammatory cytokines including IL-6, COX-2, and TNF-α.AA-induced NLPR3 infammasome cleavage can be inhibited by increasing protein p62 and activating the Nrf2 antioxidant pathway [33,111].

Acrylamide Induces Pyroptosis in Liver, Kidney, and Lung
Cells.Excessive pyroptosis was also reported in AA-induced hepatotoxicity and nephrotoxicity in rats.Not only the obvious characteristics of apoptosis and autophagy indexes but also infammatory parameters such as IL-1β, IL-6, TNFα, and COX-2 were signifcantly up-regulated in the liver and kidney of AA-treated rats [91].AA could induce pyroptosis in rat liver kupfer cell, as indicated by NLRP3 infammatory activation and increased levels of caspase-1, IL-1β, IL-18, IL-6, and TNF-α [112].It was shown that AAinduced-infammatory responses could cause pulmonary dysfunction and increase systemic infammation.Specifcally, AA resulted in signifcantly elevated levels of NF-κB, IL-1β, TNF-α, and COX-2 in lung tissue [94].Tese evidence suggested that pyroptosis represents as one of the crucial mechanisms of AA toxicity.
Pyroptosis in which cells constantly expand until the cell membrane bursts, results in the release of cell contents and activation of infammatory responses.Pyroptosis is a PCD mediated by GSDMD.AA-induced diferent degrees of pyroptosis in the nervous system, liver, kidney, and lung cells (Figure 5).NLRP3 infammasome and NF-κB have been reported to be involved in AA induced-injury.However, other specifc pathways of pyroptosis regarding to AA still need further research.

Acrylamide Induces Cell Necrosis
Te term "necrosis" (or "oncosis") refers to all forms of death that are characterized by swelling of the cells and their organelles, followed by permeabilization of the cellular membranes.Traditionally, cell necrosis is considered to be a kind of cell death induced by extreme physical and chemical factors.However, recent studies have shown that cell necrosis is not completely uncontrolled.Necrosis in some cases may be regulated, which is called necroptosis [20,22].Like pyroptosis, necroptosis also destroys cell membranes and causes a severe infammatory response.Unlike pyroptosis, necroptosis has its own executive protein diferent from that of pyroptosis.
Necroptosis is generally mediated by receptorinteracting protein kinase-1/-3 (RIP1/RIP3) and performed through MLKL (mixed-lineage kinase domainlike protein).RIP1 and RIP3 through RHIM (RIP homotypic interaction motif ) form complex IIb, also known as necrosome which mediates necroptosis [113].Mutual phosphorylation of RIP1 and RIP3 in the necrosome can lead to MLKL recruitment and phosphorylation of MLKL.Journal of Food Quality Ten, phosphorylated MLKL oligomerizes and transfers to the cell membrane, followed by destroying the permeability and integrity of the membrane, thus fnally to necroptosis.However, necroptosis is more precisely defned as a RIP3-dependent cell death, because RIP3 is essential and RIP1 is not always involved in signal transduction (Figure 6).
It has been reported that a high concentration of AA stimulated the production of reactive oxygen species and induced apoptosis and necrosis in hippocampal neurons and pluripotent neural precursor cells in mice [54].In mussels exposed to AA, female gonads endured severe necrosis and oocyte atresia [114].In rats exposed to AA, there was obvious infammatory cell infltration, hepatocellular necrosis, and hemorrhage areas in liver sections, and necrosis and glial cell activation in nervous tissues [115,116].Tese fndings only suggested the occurrence of necrosis and infammatory response in AA-induced toxicity.However, there is no direct evidence that the necrosis is some kind of   Journal of Food Quality necroptosis.In addition, the signal pathway of necroptosis has not been fully understood.Further research regarding to the mechanisms of necroptosis and AA-induced necrosis is still needed.

Summary
AA can induce varied degrees of PCD in various types cells.At present, plentiful research focuses on nervous, reproductive, liver, and kidney injuries.However, an increasing number of studies are expanding to other organs and systems in animals and humans (Figure 7).Inducing the disorder of the redox system is common and important for AA to exert toxic efects.Mitochondrial stress and endoplasmic reticulum stress, mediated by some kinases, are responsible for AA-induced PCD.Bioactive components to some degree ease the damage caused by AA.Although apoptosis, autophagy, and pyroptosis were all detected after AA exposure, existing studies hardly elucidate the interaction between them.Other types of PCD, such as mitotic disorder, are hardly reported and need further exploration.

Figure 1 :
Figure 1: Major source of exposure to AA. Tere are four main routes of exposure to AA: dietary way, occupational exposure, cosmetics, and smoking.

Figure 2 :
Figure 2: Mechanism of AA formation in heated foods.(a) Principle of decarboxylation of Schif base to form AA. (b) Te principle that dicarbonyl compounds are degraded by Strecker to form AA.

Figure 3 :
Figure 3: Molecular mechanism of apoptosis pathway induced by AA.

Figure 7 :
Figure 7: AA can induce PCD in diferent cell types.Te picture shows roughly four types of PCD.

Table 1 :
Contents of AA in diferent food categories.