Upstream eventCell 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.||adjacent||Moderate|
|Acetylcholinesterase Inhibition Leading to Neurodegeneration||adjacent||High|
Life Stage Applicability
Key Event Relationship Description
Evidence Supporting this KER
There is well established mechanistic understanding supporting the relationship between these two KEs.
Neurodegeneration in the strict sense of the word, is referring to any pathological condition primarily affecting brain cell populations (Przedborski et al., 2003). At histopathological level, neurodegenerative conditions are described by neuronal death and reactive gliosis (Przedborski et al., 2003).
Include consideration of temporal concordance here
- Acute brain damage induced by DomA is characterized by neurodegenerative changes consisting of neuronal shrinkage, vacuolization of the cytoplasm, cell drop out, edema, microvacuolation of the neuropil and hydropic cytoplasmic swelling of resident astrocytes (reviewed in Pulido et al., 2008). These histopathological changes can be identified within structures of the limbic system, in hippocampus, in the CA3, CA4 or hilus of the dentate gyrus (DG) (reviewed in Pulido et al., 2008). Other brain areas known to be affected by DomA include: the olfactory bulb, the piriform and entorhinal cortices, the lateral septum, the subiculum, the arcuate nucleus and several amygdaloid nuclei. The area postrema is another target for DomA toxicity as it has been identified in both rodents and non-human primates, providing a possible explanation of emetic symptoms (nausea, retching, and/or vomiting) induced by DomA. There has been an effort to map and create a 3-D reconstruction of DomA-induced neurodegeneration in the mouse brain demonstrating that the affected areas include the olfactory bulb, septal areas and the limbic system (Colman et al., 2005; Barlow et al., 2004).
- Female Sprague-Dawley rats dosed once intraperitoneally (i.p.) with 0, 1, 2, 4, or 7.5 DomA mg /kg of body weight have been euthanized after 24 h and their nervous system has been examined for microscopic alterations revealing neuronal degeneration and vacuolation of the neurophil in the limbic and the olfactory systems (Tryphonas et al., 1990).
- The mean of TUNEL positive cells in the hippocampus is increased (6 fold) in mice injected intraperitoneally (i.p.) at a dose of 2 DomA mg/kg once a day for 3 weeks (Lu et al., 2012). However, the same treatment protocol does not cause any neurodegeneration (Lu et al., 2012). In contrast, when the same treatment has been prolonged for one more week (total 4 weeks), the mean values of NeuN-positive cells in the hippocampal CA1 sections of DomA-treated cells decreases by 3 fold compared to controls (Lu et al., 2012). This study shows that the incidence of upstream KE (cell death) is higher than the incidence of downstream KE (neurodegeneration) and that upstream KE (cell death) precedes downstream KE (neurodegeneration).
- The bcl-2 and bax mRNA levels in the hippocampus are significantly increased at 16 h and gradually decreased at 24 h following the administration of DomA (0.75 mg/kg body weight) in adult rats. In situ hybridization analysis reveals complete loss of bcl-2, bax, and caspase-3 mRNA at 24 h after DA administration in the region of the hippocampus, whereas neurodegeneration by Nissl staining is detected at the same time point but has been reported to be more pronounced after 5 days (Ananth et al., 2001). This study demonstrates that both KEs occur after exposure to the same dose of DomA and that the upstream KE (cell death) occurs earlier than the downstream KE (neurodegeneration).
- Adult rats received i.p. injections with DomA 1.0 mg/kg/h until animals exhibited first motor seizures. After a week of recovery, aggressive behaviors and motor seizures of the animals have been monitored for 3h twice a week. After 12 weeks, animals were euthanized and brains have been examined for indications of cell loss by using thionine (Nissl) staining, which highlights the cell bodies of all living neurons. In piriform cortex a reduced cell density has been noted in the medial layer 3 (1.3-1.8 fold decrease compared to controls), an area that shows also prominent amino cupric staining (stain that assesses neuronal damage) (Tiedeken and Ramsdell, 2013a). The same research group has reported that by following the above experimental procedure but sacrificing the rats 7 days after DomA-induced seizures intense and widespread silver reaction product in the olfactory bulb occurs, whereas minor or no evident damage is found in the hippocampus (Tiedeken et al., 2013b).
- Injection of DomA 0.5 mg/kg, i.p. to adult C57BL/6 male mice resultes in loss of 32% and 30% of Nissl-stained neurons in hilus and CA1 pyramidal layer of the hippocampus, respectively, compared to control mice when they are sacrificed 7 d after the administration (Antequera et al., 2012).
- The severity and extent of hippocampal neuronal degeneration varies significantly depending on the dose of DomA (1 μM to 1 mM) that is tested after microinjection to adult male Sprague Dawley rats (Qiu and Currás-Collazo, 2006). In rats dosed with 1 mM DomA and sacrificed after 24 h, histopathological analysis using toluidine blue staining has revealed extensive neuronal damage throughout the ipsilateral hippocampal structure. Shrunken, disorganized and densely stained neurons of irregular shape have been identified throughout CA1, CA2, CA3 pyramidal layer as well as the dentate gyrus hilus and granule cells layer. For the 100 μM group animals, CA1 neuronal changes have been less prominent, whereas 10 μM and 1 μM DomA have not produced any resolvable histopathological changes (Qiu and Currás-Collazo, 2006).
- Adult male rats treated with 2 mg/kg DomA i.p. have been sacrificed after 3 d and showed that the silver stain that is used to assess neurodegeneration clearly distinguishes treated from control animals, whereas a number of other markers has failed to do so (Scallet et al., 2005). The same results have been found after even longer exposure times (7 d) to DomA (Appel et al., 1997).
- Male Wistar rats have been given a single i.v. injection of DA (0.75 mg/kg) in the right external jugular vein and brain sections have been stained with Nissl stain at 5 d after DomA administration. Histopathological analysis has revealed a large number of darkly stained shrunken neurons in the hippocampus (Ananth et al., 2003). However, complete absence of hippocampal neurons has been observed in CA1 and CA3 regions in DomA treated animals at 3 months after DomA administration (Ananth et al., 2003).
- In 2-3 week old hippocampal slice cultures, derived from 7 day old rat pups, DomA (0.1-100 µM) has been added to the culture medium and neurodegeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields has been measured. The CA1 region appears to be most sensitive to DomA, with an EC50 value of 6 µM DomA after estimating the PI-uptake at 72 h (Jakobsen et al., 2002).
- Cynomolgus monkeys have been given i.v. a range of DomA doses from 0.25 to 4.0 mg/kg. Silver staining of brain sections have revealed that doses in the range of 0.5-1.0 mg/kg produces a small area of silver grains restricted to axons of the hippocampal CA2 stratum lucidum, whereas higher concentrations produce degenerating axons and cell bodies (Slikker et al., 1998). The same research group treated i.v. adult monkeys with DomA at one of a range of doses from 0.25 to 4 mg/kg. After a week, silver staining has demonstrated degenerating axons and cell bodies that is mild and restricted to CA2 stratum lucidum at a lower doses (0.5 to 1.0 DomA mg/kg). Doses of more than 1.0 mg/kg cause widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfields of the hippocampus. However, when DomA is orally administered to cynomolgus monkeys at doses of 0.5 mg/kg for 15 days and then at 0.75 mg/kg for another 15 days no histopathoogical changes in the brain are detected (Truelove et al., 1997).
- In humans, autopsy of individuals intoxicated by DomA reveal brain damage characterized by neuronal necrosis and in the hippocampus and the amygdaloid nucleus (Pulido, 2008). The thalamus and subfrontal cortex are damaged only in some patients suffering from Amnesic Shellfish Poisoning (ASP). The detailed examination of one patient intoxicated by DomA has revealed complete neuronal loss in the CA1, CA3 and CA4 regions, whereas moderate loss is seen in the CA2 region (Cendes et al., 1995). Non-severe neuronal loss has been detected in amygdale, overlying cortex, the dorsal and ventral septal nuclei, the secondary olfactory areas, and the nucleus accumbens (Cendes et al., 1995).
|Stressor||Experimental Model||Tested concentrations||Exposure route||Exposure duration||Cell death (KE up) (measurements, quantitative if available)||Neurodegeneration (KE down) (measurements, quantitative if available)||References||Temporal Relationship||Dose-response relationship||Incidence||Comments|
|DomA||Female Sprague-Dawley rats||0, 1, 2, 4, or 7.5 DomA mg /kg||intraperitoneally (i.p.)||Euthanized after 24 h||Neuronal degeneration and vacuolation of the neuropil in the limbic and the olfactory systems||Tryphonas et al., 1990|
|DomA||16-month-old male ICR mice||2 mg/kg||Intraperitoneally (i.p.)||Once a day for 3 or 4 weeks||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.||The mean OD of NeuN immunoreactivity in the hippocampus of mice decreased (3 fold) indicating significant neuron loss by apoptosis, which is one of the pathological hallmarks of neurodegeneration||Lu et al., 2012||Upstream KE (cell death) precedes downstream KE (neurodegeneration)||Same dose||Incidence of upstream KE (cell death) is higher than the incidence of downsteam KE (neurodegeneration)||Mice treated with DomA once a day for 3 weeks showed that apoptosis was increased. However, the same treatment protocol did not cause any neurodegeneration. In contrast, when the same treatment has been prolonged for one more week (total 4 weeks) induced marked neuron loss.|
|DomA||Adult rats||0.75 mg/kg||intravenously (i.v.)||Euthanized after 2, 5, 14, or 21 days||The bcl-2 and bax mRNA levels in the hippocampus were significantly increased at 16 h and gradually decreased at 24 h following the administration of DomA. In situ hybridization analysis revealed complete loss of bcl-2, bax, and caspase-3 mRNA at 24 h after DomA administration in the region of hippocampus.||Neurodegeneration by Nissl staining was detected at the same time point but was reported to be more pronounced after 5 days||Ananth et al., 2001||Upstream KE (cell death) occurs earlier that downstream KE (neurodegeneration).||Same dose|
|DomA||Adult rats||1.0 mg/kg/h until animals exhibited first motor seizures||i.p.||Euthanized after 12 weeks||In piriform cortex a reduced cell density was noted in the medial layer 3 (1.3-1.8 fold decrease compared to controls), an area that showed also prominent amino cupric staining (stain that assesses neuronal damage).||Tiedeken and Ramsdell, 2013a|
|DomA||Adult rats||1.0 mg/kg/h until animals exhibited first motor seizures||i.p.||Euthanized after 1 week||Intense and widespread silver reaction product in the olfactory bulb, whereas minor or no evident damage was found in hippocampus.||Tiedeken et al., 2013b|
|DomA||Adult C57BL/6 male mice||0.5 mg/kg||i.p.||Euthanized after 1 week||DomA treatment resulted in the loss of 32% and 30% of Nissl-stained neurons in hilus and CA1 pyramidal layer of the hippocampus, respectively, compared to control mice.||Antequera et al., 2012|
|DomA||Adult male Sprague Dawley rats||1 μM to 1 mM||microinjection||Euthanized after 24 h||In rats dosed with 1 mM DomA and sacrificed after 24 h, histopathological analysis using toluidine blue staining revealed extensive neuronal damage throughout the ipsilateral hippocampal structure. Shrunken, disorganized and densely stained neurons of irregular shape were identified throughout CA1, CA2, CA3 pyramidal layer as well as the dentate gyrus hilus and granule cells layer. For the 100 μM group animals, CA1 neuronal changes were less prominent, whereas 10 μM and 1 μM DomA did not produce resolvable histopathological changes.||Qiu and Currás-Collazo, 2006|
|DomA||Adult male rats||2 mg/kg||i.p.||Euthanized after 3 or 7 days||DA treatment for 3 d showed that the silver stain that was used to assess neurodegeneration clearly distinguished treated from control animals , the same was true for longer exposure time (7 d).||Scallet et al., 2005, Appel et al., 1997|
|DomA||Male Wistar rats||0.75 mg/kg||i.v.||Euthanized after 5 days or 3 months||Histopathological analysis revealed a large number of darkly stained shrunken neurons in the hippocampus However, complete absence of hippocampal neurons was observed in CA1 and CA3 regions in DA treated animals at 3 months after DomA administration.||Ananth et al., 2003|
|DomA||2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups||0.1-100 µM||72 h||DomA induced neurodegeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields. The CA1 region appeared to be most sensitive to DomA, with an EC50 value of 6 µM DomA, estimated from the PI-uptake at 72 h .||Jakobsen et al., 2002|
|DomA||Cynomolgus monkeys||0.25 to 4.0 mg/kg||i.v.||Euthanized after 1 week||Silver staining of brain sections revealed that doses in the range of 0.5-1.0 mg/kg produce a small area of silver grains restricted to axons of the hippocampal CA2 stratum lucidum, whereas higher concentrations revealed degenerating axons and cell bodies. After a week, silver staining demonstrated degenerating axons and cell bodies that was mild and restricted to CA2 stratum lucidum at the lower doses (0.5 to 1.0 DomA mg/kg). Doses of more than 1.0 mg/kg caused widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfields of the hippocampus.||Slikker et al., 1998, Truelove et al., 1997|
Gap of knowledge: there are no studies showing that GLF-induced cell death leads to neurodegeneration.
Uncertainties and Inconsistencies
Zebrafish has been exposed for 36-weeks to DomA and has showed no excitotoxic neuronal death and no histopathological lesions in glutamate-rich brain areas (Hiolski et al., 2014).
Administration of DomA (9.0 mg DomA kg(-1) bw, i.p.) to Sparus aurata (seabream) leads to measurement of 0.61, 0.96, and 0.36 mg DomA kg(-1) of brain tissue at 1, 2 and 4 hours. At this dose but also at lower concentrations (0.45 and 0.9 mg DomA kg(-1) bw) no major permanent brain damage has been detected (Nogueira et al., 2010). Leopard sharks possess the molecular target for DomA but it has been shown to be resistant to doses of DomA that can cause neurotoxicity to other vertebrates, suggesting the presence of some protective mechanism (Schaffer et al., 2006).
All these reports support the view that there is possible a species specific susceptibility to DomA toxicity.
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 KERs are described in the table above.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
There is an overall agreement regarding the histopathology of the brain lesions related to acute DomA neurotoxicity across certain species. Data derived from humans, rodents, non-human primates and sea lions suggest that common neudegeneration features in selected brain areas are found despite the fact that study design, estimated exposure, processing of samples and history of event may differ (Pulido, 2008).
Furthermore, the distribution of brain damage by DomA has also been established by magnetic resonance imaging microscopy (MRM) for both human and rat, demonstrating similar distribution as that described by histopathological studies (Pulido, 2008).
It is important to notice that human sensitivity to DomA exposure is well documented in the published literature and seems to be much higher than in other species (Lefebvre and Robertson 210; Barlow et al., 2004).In 1987 in Canada, more than 200 people became acutely ill after ingesting of mussels contaminated with DomA. The outbreak resulted in 20 hospitalizations and four deaths. Clinical effects observed included gastrointestinal symptoms and neurotoxic effects such as hallucinations, memory loss and coma. For this reason, the condition was termed amnesic shellfish poisoning (Barlow et al., 2004). The neurotoxic properties of domoic acid result in neuronal degeneration and necrosis in specific regions of the hippocampus (Teitelbaum et al., 1990).
Ananth C, Thameem DS, Gopalakrishnakone P, Kaur C., Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response. J Neurosci Res., 2001, 66: 177-190.
Ananth C, Gopalakrishnakone P, Kaur C., Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus. Neurosci Lett., 2003, 338: 49-52.
Antequera D, Bolos M, Spuch C, Pascual C, Ferrer I, Fernandez-Bachiller MI, Rodríguez-Franco MI, Carro E., Effects of a tacrine-8-hydroxyquinoline hybrid (IQM-622) on Aβ accumulation and cell death: involvement in hippocampal neuronal loss in Alzheimer's disease. Neurobiol Dis., 2012, 46: 682-691.
Appel NM, Rapoport SI, O'Callaghan JP., Sequelae of parenteral domoic acid administration in rats: comparison of effects on different anatomical markers in brain. Synapse, 1997, 25: 350-358.
Barlow Jeffery B, T, Moizer K, Paul S, and Boyle C., Amnesic shellfish poison. Food Chem Toxicol., 42: 545-557.
Cendes F, Andermann F, Carpenter S, Zatorre RJ, Cashman NR., Temporal lobe epilepsy caused by domoic acid intoxication: evidence for glutamate receptor-mediated excitotoxicity in humans. Ann Neurol., 1995, 37: 123-6.
Colman JR, Nowocin KJ, Switzer RC, Trusk TC, Ramsdell JS., Mapping and reconstruction of domoic acid-induced neurodegeneration in the mouse brain. Neurotoxicol Teratol., 2005, 27: 753-767.
Hiolski EM, Kendrick PS, Frame ER, Myers MS, Bammler TK, Beyer RP, Farin FM, Wilkerson HW, Smith DR, Marcinek DJ, Lefebvre KA., Chronic low-level domoic acid exposure alters gene transcription and impairs mitochondrial function in the CNS. Aquat Toxicol., 2014, 155: 151-159.
Jakobsen B, Tasker A, Zimmer J., Domoic acid neurotoxicity in hippocampal slice cultures. Amino Acids, 2002, 23: 37-44.
Lefebvre Kathi A. and Robertson Alison, Domoic acid and human exposure risks: A review, Toxicon, 2010, 56: 218–230.
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: 646-659.
Nogueira I, Lobo-da-Cunha A, Afonso A, Rivera S, Azevedo J, Monteiro R, Cervantes R, Gago-Martinez A, Vasconcelos V., Toxic effects of domoic acid in the seabream Sparus aurata. Mar Drugs, 2010, 8: 2721-2732.
Przedborski S, Vila M, Jackson-Lewis V., Neurodegeneration: What is it and where are we? J Clin Invest., 2003, 111: 3-10.
Pulido OM., Domoic acid toxicologic pathology: a review. Mar Drugs, 2008, 6: 180-219.
Qiu S, Currás-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol., 2006, 28: 354-362.
Scallet AC, Schmued LC., Johannessen JN. Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid. Neurotoxicol Teratol., 2005, 27: 745-752.
Schaffer P, Reeves C, Casper DR, Davis CR., Absence of neurotoxic effects in leopard sharks, Triakis semifasciata, following domoic acid exposure. Toxicon., 2006, 47: 747-752.
Slikker W Jr, Scallet AC, Gaylor DW., Biologically-based dose-response model for neurotoxicity risk assessment. Toxicol Lett., 1998, 102-103: 429-433.
Teitelbaum JS, Zatorre RJ, Carpenter S, Gendron D, Evans AC, Gjedde A, and Cashman NR., Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med., 1990, 322: 1781-1787.
Tiedeken JA, Muha N, Ramsdell JS., A cupric silver histochemical analysis of domoic acid damage to olfactory pathways following status epilepticus in a rat model for chronic recurrent spontaneous seizures and aggressive behavior. Toxicol Pathol., 2013a, 41: 454-69.
Tiedeken JA, Ramsdell JS., Persistent neurological damage associated with spontaneous recurrent seizures and atypical aggressive behavior of domoic acid epileptic disease. Toxicol Sci., 2013b, 133: 133-43.
Truelove J, Mueller R, Pulido O, Martin L, Fernie S, Iverson F., 30-day oral toxicity study of domoic acid in cynomolgus monkeys: lack of overt toxicity at doses approaching the acute toxic dose. Nat Toxins., 1997, 5: 111-114.
Tryphonas L, Truelove J, Nera E, Iverson F., Acute neurotoxicity of domoic acid in the rat. Toxicol Pathol., 1990, 18: 1-9.