Upstream eventN/A, Mitochondrial dysfunction 1
N/A, Cell injury/death
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.||non-adjacent||Moderate|
|Endocytic lysosomal uptake leading to liver fibrosis||adjacent||Moderate||Low|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
ROS generation is known to activate different pathways leading to apoptosis, whereas depletion of energy production induces necrotic cell death.
Evidence Supporting this KER
There is functional mechanistic understanding supporting this relationship between KE3 and KE4.
ROS are known to stimulate a number of events and pathways that lead to apoptosis, triggered by ROS-induced ER stress signalling pathway (Lu et al., 2014), caspase-dependent and -independent apoptosis (Zhou et al., 2015), mitogen-activated protein kinase (MAPK) signal transduction pathways (reviewed in Cuadrado and Nebreda, 2010, Harper and LoGrasso, 2001).
Depletion of cellular ATP is known to cause switching from apoptotic cell death triggered by a variety of stimuli to necrotic cell death (Leist et al., 1997) suggesting that the level of intracellular ATP determines whether the cell dies by apoptosis or necrosis (Nicotera et al., 1998). There is strong proof that apoptosis requires energy, as it is a highly regulated process involving a number of ATP-dependent steps such as caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, bleb formation and apoptotic body formation (Richter et al., 1996).
Include consideration of temporal concordance here
In the case of DomA, in vitro studies have shown that oxidative stress and oxidative stress-induced activation of the stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK) pathway is implicated in DomA-mediated apoptosis (Giordano et al., 2007; 2008; 2009; Lu et al., 2010). In vivo findings also show that ROS-mediated cognitive deficits are associated with apoptosis induced by activation of the JNK pathway (Lu et al., 2010; 2011).
- Mice injected intraperitoneally (i.p.) with DomA at a dose of 2 mg/kg once a day for 4 weeks have shown increase (6 fold) of the TUNEL positive cells in the hippocampus . In the same study they have found that indicators of mitochondria function are markedly decreased (1.5-2 fold) and ROS levels are elevated (3.2 fold) (Lu et al., 2012). DomA treatment also significantly decreases the levels of bcl-2, procaspase-3 and procaspase-12 and increases the activation of caspase-3 and caspase-12 in the mouse hippocampus (Lu et al., 2012). The same research group using similar dose but longer exposure (4 weeks), has shown increase of ROS (3 fold) and NOX (2 fold) and elevated (8 fold) mean value of TUNEL-positive cells in the hippocampal CA1 sections as well as increase in the activation of caspase-8 and caspase-3 (Wu et al., 2012). These two in vivo studies (Lu et al., 2012; Wu et al., 2012) suggest that both KEs are affected in response to the same dose of DomA and exposure paradigm and that the incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction).
- The cell viability has been measured by the MTT reduction assay in mouse cerebellar granule neurons (CGNs) and showed that the IC50 values for DomA are 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons (Giordano et al., 2006). This reduction in cell viability has been demonstrated to be concentration dependent after studying a range of concentrations of DomA (0.01 and 10 µM). Giordano et al. 2007 have shown that 100 nM DomA induce apoptotic cell death in mouse CGNs. In a follow-up study, the same research group has performed a dose response evaluation and showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs derived from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione are more sensitive as 10 nM DomA induces a significant increase in apoptotic cell number (Giordano et al., 2009). The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes has been caused by 100 nM DomA. Interestingly, 1 and 10 µM DomA still cause significant apoptosis in both cell types but to a lesser extent compared to 100 nM DomA. ROS have been measured only at the dose of 100 nM DomA, 30 min after treatment and showed 2.5 fold increase compared to controls in CGNs from Gclm (+/+) mice (Giordano et al., 2009). Caspase 3 activity has also been measured after 12 h with prior 1 h exposure to 100 nM DomA and found to be increased (2.2 fold). In the same study, DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h exposure. Again these in vitro studies (Giordano et al., 2007; 2009) suggest that both KEs are affected by the same dose of DomA and that the incidence of KE down (cell death) is higher than the incidence of KE up (mitochondrial dysfunction). Furthermore, KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that takes place 12-24 h later.
- Mixed cortical cultures have been treated with 3, 5, 10, or 50 μM DomA for a variety of exposure durations (10 min, 30 min, 1 h, or 2 h), after which DomA is washed out and the culture medium is replaced with conditioned medium from unexposed sister cultures (Qiu et al., 2006). In all cases neuronal death has been measured 24 h following the beginning of exposure. The results show that DomA-induced neuronal death is determined by both concentration and duration of exposure. After a 10-min exposure, 50 μM DomA produces marked neuronal death of 47.4 %, whereas by 1 h of treatment, the same concentration produces near maximal neuronal death but longer exposures do not increase neuronal death further (Qiu et al., 2006). Regarding time dependence, this study shows that low concentrations of DomA produces more neuronal death if this is measured 22 h after the washout than if measured immediately after DomA treatment, while higher concentrations of DomA (20–100 μM) produces equivalent degrees of neuronal death when measured at these two time points (Qiu et al., 2006). Based on these findings, three EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration of exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) (Qiu et al., 2006).
- The mean concentration of DomA in rat brain samples obtained at 30 min after intraperitoneal (i.p.) administration of 1 mg/kg DA is 7.2 ng/g (Tsunekawa et al., 2013). These animals have been examined and revealed after histopathological analysis neuronal shrinkage and cell death, including an increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and after 5 days (19.0 %) compared to the controls (1.7 %) (Tsunekawa et al., 2013). In the same study, indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone (Tsunekawa et al., 2013).
- Brain slices from 8-day-old pups have been treated after 2 weeks with 10 μM DomA and assessed with propidium iodine (PI) stain to determine cellular damage (Erin and Billingsley, 2004). A time course has been carried out and viable cultures have been visualized 12, 24, 48 and 92 h after DomA treatment. Changes in PI uptake has been detected after 24 h post-treatment and at 4h the average fold-increase of PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively (Erin and Billingsley, 2004). In the same study, incubation of brain slices with DomA induces degradation of α-spectrin to the 120-kDa product after 18 h of treatment but no change has been noted after 12 h incubation, whereas caspase 3 activity results have not been conclusive (Erin and Billingsley, 2004).
- Using observations of neuronal viability and morphology, exposure of cultured murine cortical neurones to DomA for 24 h have shown to induce concentration-dependent neuronal cell death and the EC50 determined to be 75 µM (Larm et al., 1997).
|Stressor||Experimental Model||Tested concentrations||Exposure route||Exposure duration||Mitochondrial dysfunction (KE up) (measurements, quantitative if available)||Cell death (KE down) (measurements, quantitative if available)||References||Temporal Relationship||Dose-response relationship||Incidence||Comments|
|DomA||16-month-old male ICR mice||2 mg/kg||Intraperitoneally (i.p.)||Once a day for 4 weeks||Indicators of mitochondrial function were markedly decreased (1.5-2 fold) and ROS levels were elevated (3.2 fold).||The mean of TUNEL positive cells in the hippocampus was increased (6 fold). The levels of bcl-2, procaspase-3 and procaspase-12 were significantly decreased and the activation of caspase-3 and caspase-12 in the mouse hippocampus were increased.||Lu et al., 2012||Same dose||Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)|
|DomA||16-month-old male ICR mice||2 mg/kg||i.p.||Once a day for 4 weeks||ROS levels were increased (3 fold) and NOX (2 fold).||The mean value of TUNEL-positive cells in the hippocampal CA1 sections was elevated (8 fold) and the activation of caspase-8 and caspase-3 was increased.||Wu et al., 2012||Same dose||Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)|
|DomA||Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice||0.01 to 10 µM||Time course (15 to 120 min)||DomA caused a significant time- and concentration-dependent increase in ROS production. The higher ROS production (2.5 fold increase) was recorded after 1 h of exposure.||IC50 values for DomA were 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons based on MTT assay after 24 h of exposure.||Giordano et al., 2006||KE up (mitochondrial dysfunction) happens earlier than KE down (cell death)||Same doses|
|DomA||CGNs from Gclm (+/+) and Gclm (−/−) mice||0.01 to 10 µM||Time course (0 to 180 min)||DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.||0.1μM DomA was maximally effective in inducing apoptosis, while a concentration causing high toxicity (10μM) induced very limited apoptosis, 24 h after exposure.||Giordano et al., 2007||KE up (mitochondrial dysfunction) happens earlier (1-2 h) than KE down (cell death) that occurs after 24 h||Same doses|
|DomA||CGNs from Gclm (+/+) and Gclm (−/−) mice||0.01 to 10 µM||For ROS: 30min, Apoptosis: 12-24 h.||ROS levels were measured only at the dose of 100 nM DomA 30 min after treatment in CGNs from Gclm (+/+) mice and showed 2.5 fold increase compared to controls .||A dose response study that showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione were more sensitive as 10 nM DomA induced a significant increase in apoptotic cells number .The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes was caused by 100 nM DomA. 1 and 10 µM DA caused significant apoptosis in both cell types but to less extend compared to 100 nM DomA. Caspase 3 activity after 12 h with prior 1 h exposure to 100 nM DomA found to be increased (2.2 fold). DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h from exposure.||Giordano et al., 2009||KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that take place 12-24 h later||Same dose||Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)|
|DomA||Mixed cortical cultures obtained from pregnant Holtzman rats on embryonic day (ED) 16–18||3, 5, 10, or 50 μM||10 min, 30 min, 1 h or 2 h, after which DomA was washed out and the culture medium replaced with conditioned medium from unexposed sister cultures .||EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) .||Qiu et al., 2006|
|DomA||Rat||1 mg/kg DA||i.p.||Indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone .||Neuronal shrinkage and cell drop out as well as increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and 5 days (19.0 %) has been found compared with that of controls (1.7 %) .||Tsunekawa et al., 2013|
|DomA||Rat rain slices from 8-day-old pups||10 μM||Time course (12, 24, 48 and 92 h) after DomA treatment.||PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively . Degradation of α-spectrin to the 120-kDa product after 18 h of DomA treatment was noted but no change was noted after 12 h incubation, whereas caspase 3 activity results were not conclusive.||Erin and Billingsley, 2004|
|DomA||Cultured murine cortical neurones||DomA induces concentration-dependent neuronal cell death and the EC50 determined to be 75 µM .||Larm et al., 1997|
Gap of knowledge: there are no studies showing that GLF induces neuronal cell death through mitochondrial dysfunction.
Uncertainties and Inconsistencies
Rats have been administered with DA at the dose of 1.0 mg/kg for 15 days. The histochemical analysis of hippocampus from these animals has revealed no presence of apoptotic bodies and no Fluoro-Jade B positive cells (Schwarz et al., 2014).
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
The experiments describing semi-quantitative effects for this KER is described in the table above.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Neuronal necrosis has been noted in sea lions accidentally exposed to DomA (Silvagni et al., 2005) that correlated well with the histopathological findings previously reported in experimental studies (Tryphonas et al., 1990).
Cuadrado A, Nebreda AR., Mechanisms and functions of p38 MAPK signalling. Biochem J., 2010, 429(3): 403–417.
Erin N, Billingsley ML., Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices. Brain Res., 2004, 1014: 45-52.
Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70: 2116-2126.
Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.
Giordano G, Klintworth HM, Kavanagh TJ, Costa LG., Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases. Neurochem Int., 2008, 52: 1100-1105.
Giordano G, Li L, White CC, Farin FM, Wilkerson HW, Kavanagh TJ, Costa LG., Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells. J Neurochem., 2009, 109: 525-538.
Harper SJ, LoGrasso P., Signalling for survival and death in neurones: the role of stress-activated kinases. JNK and p38. Cell Signal., 2001, 13(5): 299–310.
Larm JA, Beart PM, Cheung NS., Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones. Neurochem Int., 1997, 31: 677-682.
Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P., Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med., 1997, 185: 1481−1486.
Lu J, Wu DM, Zheng YL, Hu B, Zhang ZF., Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol., 2010, 20: 598-612.
Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B, Zhang ZF., Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain., 2011, 134: 783-797.
Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52(3): 646-59.
Lu TH, Su CC, Tang FC, Chen CH, Yen CC, Fang KM, Lee KL, Hung DZ, Chen YW., Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway. Chem Biol Interact., 2014, 225: 1-12.
Nicotera P, Leist M, Ferrando-May E., Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicol Lett., 1998, 102-103: 139-142.
Qiu S, Pak CW, Currás-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical cultures: effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006, 89: 243-256.
Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett., 1996, 378: 107-110.
Schwarz M, Jandová K, Struk I, Marešová D, Pokorný J, Riljak V. Low dose domoic acid influences spontaneous behavior in adult rats. Physiol Res., 2014, 63: 369-76.
Silvagni PA, Lowenstine LJ, Spraker T, Lipscomb TP, Gulland FMD., Pathology of Domoic Acid Toxicity in California Sea Lions (Zalophus californianus). Vet Path., 2005, 42: 184-191.
Tryphonas L, Truelove J, Iverson F, Todd EC, Nera EA. Neuropathology of experimental domoic acid poisoning in non-human primates and rats. Can Dis Wkly Rep. 1990 Sep;16 Suppl 1E:75-81.
Tsunekawa K, Kondo F, Okada T, Feng GG, Huang L, Ishikawa N, Okada S., Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation. BMC Neurosci., 2013, 14: 4-16.
Wu DM, Lu J, Zheng YL, Zhang YQ, Hu B, Cheng W, Zhang ZF, Li MQ., Small interfering RNA-mediated knockdown of protein kinase C zeta attenuates domoic acid-induced cognitive deficits in mice. Toxicol Sci., 2012, 128: 209-222.
Zhou Q, Liu C, Liu W, Zhang H, Zhang R, Liu J, Zhang J, Xu C, Liu L, Huang S, Chen L., Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis. Toxicol Sci., 2015, 143: 81-96.