Chronic Domoic Acid Research Articles

Clinical Signs and Pathology Associated With Domoic Acid Toxicosis in Southern Sea Otters (Enhydra lutris nereis)

The marine biotoxin domoic acid (DA) is an analog of the neurotransmitter glutamate that exerts potent excitatory activity in the brain, heart, and other tissues. Produced by the diatomPseudo-nitzschiaspp., DA accumulates in marine invertebrates, fish, and sediment. Southern sea otters (Enhydra lutris nereis) feed primarily on invertebrates, including crabs and bivalves, that concentrate and slowly depurate DA. Due to their high prey consumption (25% of body weight/day), sea otters are commonly exposed to DA. A total of 823 necropsied southern sea otters were examined to complete this study; first we assessed 560 subadult, adult, and aged adult southern sea otters sampled from 1998 through 2012 for DA-associated pathology, focusing mainly on the central nervous system (CNS) and cardiovascular system. We applied what was learned to an additional cohort of necropsied sea otters of all demographics (including fetuses, pups, juveniles, and otters examined after 2012:n= 263 additional animals). Key findings derived from our initial efforts were consistently observed in this more demographically diverse cohort. Finally, we assessed the chronicity of DA-associated pathology in the CNS and heart independently for 54 adult and aged adult sea otters. Our goals were to compare the temporal consistency of DA-associated CNS and cardiovascular lesions and determine whether multiple episodes of DA toxicosis could be detected on histopathology. Sea otters with acute, fatal DA toxicosis typically presented with neurological signs and severe, diffuse congestion and multifocal microscopic hemorrhages (microhemorrhages) in the brain, spinal cord, cardiovascular system, and eyes. The congestion and microhemorrhages were associated with detection of high concentrations of DA in postmortem urine or gastrointestinal content and preceded histological detection of cellular necrosis or apoptosis. Cases of chronic DA toxicosis often presented with cardiovascular pathology that was more severe than the CNS pathology; however, the lesions at both sites were relatively quiescent, reflecting previous damage. Sea otters with fatal subacute DA toxicosis exhibited concurrent CNS and cardiovascular pathology that was characterized by progressive lesion expansion and host response to DA-associated tissue damage. Acute, subacute, and chronic cases had the same lesion distribution in the CNS and heart. CNS pathology was common in the hippocampus, olfactory, entorhinal and parahippocampal cortex, periventricular neuropil, and ventricles. The circumventricular organs were identified as important DA targets; microscopic examination of the pituitary gland, area postrema, other circumventricular organs, and both eyes facilitated confirmation of acute DA toxicosis in sea otters. DA-associated histopathology was also common in cardiomyocytes and coronary arterioles, especially in the left ventricular free wall, papillary muscles, cardiac apex, and atrial free walls. Progressive cardiomyocyte loss and arteriosclerosis occurred in the same areas, suggesting a common underlying mechanism. The temporal stage of DA-associated CNS pathology matched the DA-associated cardiac pathology in 87% (n= 47/54) of cases assessed for chronicity, suggesting that the same underlying process (e.g., DA toxicosis) was the cause of these lesions. This temporally matched pattern is also indicative of a single episode of DA toxicosis. The other 13% of examined otters (n= 7/54) exhibited overlapping acute, subacute, or chronic DA pathology in the CNS and heart, suggestive of recurrent DA toxicosis. This is the first rigorous case definition to facilitate diagnosis of DA toxicosis in sea otters. Diagnosing this common but often occult condition is important for improving clinical care and assessing population-level impacts of DA exposure in this federally listed threatened subspecies. Because the most likely source of toxin is through prey consumption, and because humans, sea otters, and other animals consume the same marine foods, our efforts to characterize health effects of DA exposure in southern sea otters can provide strong collateral benefits.

Discovery of a Potential Human Serum Biomarker for Chronic Seafood Toxin Exposure Using an SPR Biosensor.

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 μg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers’ urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern.

Chronic, low-level oral exposure to marine toxin, domoic acid, alters whole brain morphometry in nonhuman primates

Domoic acid (DA) is an excitatory neurotoxin produced by marine algae and responsible for Amnesiac Shellfish Poisoning in humans. Current regulatory limits (˜0.075-0.1 mg/kg/day) protect against acute toxicity, but recent studies suggest that the chronic consumption of DA below the regulatory limit may produce subtle neurotoxicity in adults, including decrements in memory. As DA-algal blooms are increasing in both severity and frequency, we sought to better understand the effects of chronic DA exposure on reproductive and neurobehavioral endpoints in a preclinical nonhuman primate model. To this end, we initiated a long-term study using adult, female Macaca fascicularis monkeys exposed to daily, oral doses of 0.075 or 0.15 mg/kg of DA for a range of 321–381, and 346–554 days, respectively. This time period included a pre-pregnancy, pregnancy, and postpartum period. Throughout these times, trained data collectors observed intentional tremors in some exposed animals during biweekly clinical examinations. The present study explores the basis of this neurobehavioral finding with in vivo imaging techniques, including diffusion tensor magnetic resonance imaging and spectroscopy. Diffusion tensor analyses revealed that, while DA exposed macaques did not significantly differ from controls, increases in DA-related tremors were negatively correlated with fractional anisotropy, a measure of structural integrity, in the internal capsule, fornix, pons, and corpus callosum. Brain concentrations of lactate, a neurochemical closely linked with astrocytes, were also weakly, but positively associated with tremors. These findings are the first documented results suggesting that chronic oral exposure to DA at concentrations near the current human regulatory limit are related to structural and chemical changes in the adult primate brain.

Maternal‐Fetal Toxicokinetics of the Shellfish Toxin Domoic Acid

BackgroundDomoic Acid (DA) is an excitatory neurotoxin produced by harmful algal blooms. Through the marine food web, humans and marine mammals can be exposed to DA and experience DA poisoning. To protect humans, a tolerable daily intake (TDI) of 0.075 mg/kg of DA has been suggested based on acute/short term exposure “no observable adverse effect level” (NOAEL) in animals with a 10‐fold safety factor. However, several studies in model species have suggested that chronic DA exposure at subacute level is also harmful to the central nervous system. Similarly, it has been suggested that in utero exposure to DA adversely affects fetal neurological development which highlights the need to establish safe chronic consumption levels in vulnerable populations. The purpose of this study was to characterize DA disposition during pregnancy following chronic subacute dosing, and to determine whether such chronic DA exposure results in significant fetal exposures and alters infant behavioral outcomes in cynomolgus monkeys.MethodFemale monkeys (n=10) were orally dosed daily with 0.15 mg/kg DA for at least two months prior to and over the course of pregnancy. DA pharmacokinetics were characterized pre‐pregnancy, and on weeks 9 and 17 of gestation. At delivery, the maternal plasma and amniotic fluid DA concentrations were measured. Infant plasma DA concentrations were measured from birth to day 7 and neonatal behavioral outcomes were measured from birth to day 14. Maternal‐fetal pharmacokinetics of DA through pregnancy was simulated using physiologically‐based pharmacokinetic (PBPK) modeling.ResultsThe renal clearance of DA was significantly increased in these monkeys in parallel with increased GFR during pregnancy, but the overall exposure to DA was similar during pregnancy as before pregnancy (Table 1). Average maternal plasma, fetal plasma, and amniotic fluid concentrations at time of delivery were 2.1 ± 1.3 ng/mL (n=9), 1.3 ± 1.0 ng/mL (n=8), and 7.5 ± 5.0 ng/mL (n=5), respectively. Fetal to maternal plasma and amniotic fluid to maternal plasma concentration ratios were 0.6 (95% CI: 0.4, 1.0) and 3.5 (95% CI: 1.8, 7.1). Results from behavioral testing of the infants suggested that in utero exposure to DA at subacute dose levels may be associated with adverse effects on neurocognitive development.ConclusionThe results of this study suggest that mother and fetus exposures are nearly similar following repeated doses of DA during pregnancy. The high amniotic fluid to maternal concentration ratio suggests that amniotic fluid acts as a “deep compartment” of fetal distribution, and may contribute to continuous fetal exposure in utero. Collectively the data suggest that the TDI estimates for DA may need to be lowered to assure maternal‐fetal safety.Support or Funding InformationR01 ES023043‐01 NIH/NIEHSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic low-level exposure to the common seafood toxin domoic acid causes cognitive deficits in mice

The consumption of one meal of seafood containing domoic acid (DA) at levels high enough to induce seizures can cause gross histopathological lesions in hippocampal regions of the brain and permanent memory loss in humans and marine mammals. Seafood regulatory limits have been set at 20mgDA/kg shellfish to protect human consumers from symptomatic acute exposure, but the effects of repetitive low-level asymptomatic exposure remain a critical knowledge gap. Recreational and Tribal-subsistence shellfish harvesters are known to regularly consume low levels of DA. The aim of this study was to determine if chronic low-level DA exposure, at doses below those that cause overt signs of neurotoxicity, has quantifiable impacts on cognitive function. To this end, female C57BL/6NJ mice were exposed to asymptomatic doses of DA (≈0.75mg/kg) or vehicle once a week for several months. Spatial learning and memory were tested in a radial water maze paradigm at one, six and 25 weeks of exposure, after a nine-week recovery period following cessation of exposure, and at three old age time points (18, 24 and 28 months old). Mice from select time points were also tested for activity levels in a novel cage environment using a photobeam activity system. Chronic low-level DA exposure caused significant spatial learning impairment and hyperactivity after 25 weeks of exposure in the absence of visible histopathological lesions in hippocampal regions of the brain. These cognitive effects were reversible after a nine-week recovery period with no toxin exposure and recovery was sustained into old age. These findings identify a new potential health risk of chronic low-level exposure in a mammalian model. Unlike the permanent cognitive impacts of acute exposure, the chronic low-level effects observed in this study were reversible suggesting that these deficits could potentially be managed through cessation of exposure if they also occur in human seafood consumers.

Acute and chronic dietary exposure to domoic acid in recreational harvesters: A survey of shellfish consumption behavior

Domoic acid (DA) is a neurotoxin that is naturally produced by phytoplankton and accumulates in seafood during harmful algal blooms. As the prevalence of DA increases in the marine environment, there is a critical need to identify seafood consumers at risk of DA poisoning. DA exposure was estimated in recreational razor clam (Siliqua patula) harvesters to determine if exposures above current regulatory guidelines occur and/or if harvesters are chronically exposed to low levels of DA. Human consumption rates of razor clams were determined by distributing 1523 surveys to recreational razor clam harvesters in spring 2015 and winter 2016, in Washington, USA. These consumption rate data were combined with DA measurements in razor clams, collected by a state monitoring program, to estimate human DA exposure. Approximately 7% of total acute exposures calculated (including the same individuals at different times) exceeded the current regulatory reference dose (0.075mgDA·kgbodyweight−1·d−1) due to higher than previously reported consumption rates, lower bodyweights, and/or by consumption of clams at the upper range of legal DA levels (maximum 20mg·kg−1 wet weight for whole tissue). Three percent of survey respondents were potentially at risk of chronic DA exposure by consuming a minimum of 15 clams per month for at 12 consecutive months. These insights into DA consumption will provide an additional tool for razor clam fishery management.

Domoic acid-induced seizures in California sea lions (Zalophus californianus) are associated with neuroinflammatory brain injury

California sea lions (CSLs) exposed to the marine biotoxin domoic acid (DA) develop an acute or chronic toxicosis marked by seizures and act as sentinels of the disease. Experimental evidence suggests that oxidative stress and neuroinflammation are important mechanisms underlying the seizurogenic potential of environmental toxicants but these pathways are relatively unstudied in CSLs. In the current study, we investigated the role of glutamate–glutamine changes and gliosis in DA-exposed CSLs to better understand the neurotoxic mechanisms occurring during DA toxicity. Sections from archived hippocampi from control and CSLs diagnosed with DA toxicosis were immunofluorescently stained for markers of gliosis, oxidative/nitrative stress and changes in glutamine synthetase (GS). Quantitative assessment revealed increasing loss of microtubule associated protein-2 positive neurons with elevations in 4-hydroxynonenal correlating with chronicity of exposure, whereas the pattern of activated glia expressing nitric oxide synthase 2 and tumor necrosis factor followed pathological severity. There was no significant change in the amount of GS positive cells but there was increased 3-nitrotyrosine in GS expressing cells and in neurons, particularly in animals with chronic DA toxicosis. These changes were consistently seen in the dentate gyrus and in the cornu ammonis (CA) sectors CA3, CA4, and CA1. The results of this study indicate that gliosis and resultant changes in GS are likely important mechanisms in DA-induced seizure that need to be further explored as potential therapies in treating exposed wildlife.

Oxidative Stress and Redistribution of Glutamine Synthetase in California Sea Lions (Zalophus californianus) with Domoic Acid Toxicosis

The aim of this study was to test the hypothesis that oxidative stress and glutamine synthetase (GS) redistribution occur in domoic acid (DA) toxicosis in California sea lions (CSLs, Zalophus californianus). Sections of archived hippocampi from seven control and 13 CSLs diagnosed with DA toxicosis were labelled immunohistochemically for GS and for two markers of oxidative stress, malondialdehyde (MDA) and 3-nitrotyrosine (NT). The distribution and intensity of labelling were compared with the pathological changes seen in haematoxylin and eosin-stained sections. Increased expression of MDA and NT occurred in neurons of the hippocampal formation of CSLs with lesions consistent with DA toxicosis. The degree of oxidative stress was not affected significantly by the chronicity or severity of hippocampal damage. In six out of seven CSLs with chronic effects of DA toxicosis, in addition to the normal glial distribution of GS, GS expression was very strong in some neurons of the subiculum. However, neuronal GS labelling was also seen in one control CSL, an effect that may have been due to previous exposure to DA. GS expression in neurons was associated with decreases in GS labelling in neighbouring glial cell processes. DA toxicosis therefore induces increased expression of markers of oxidative stress in neurons consistent with oxidative stress contributing to the initial DA insult and also the epilepsy that often develops in chronic DA toxicosis. GS redistribution occurred primarily in chronic DA toxicosis, perhaps leading to alterations of the glutamine-glutamate-GABA (gamma-aminobutyric acid) cycle and contributing to the excitotoxicity and seizures often seen in DA toxicosis.

Evaluation of circulating eosinophil count and adrenal gland function in California sea lions naturally exposed to domoic acid

To determine the effect of natural exposure to domoic acid (DA) on eosinophil counts and adrenal gland function in California sea lions (Zalophus californianus). Cross-sectional prospective study. 39 California sea lions. Adult female sea lions admitted to a rehabilitation hospital during 2009 were classified into 1 of 3 groups (acute DA toxicosis, chronic DA toxicosis, or no DA exposure) on the basis of clinical signs, DA concentration in urine or feces, and hippocampal morphology. Endoparasite burden, eosinophil count, and serum cortisol and plasma ACTH concentrations were determined for each sea lion. For a subset of 8 sea lions, fecal glucocorticoid concentration after IM administration of cosyntropin was determined. Sea lions exposed to DA (acute DA toxicosis, n = 11; chronic DA toxicosis, 19) had higher eosinophil counts and lower serum cortisol concentrations, compared with values for sea lions with no DA exposure (9). Eosinophil count was not associated with endoparasite burden. Serum cortisol concentration was associated with plasma ACTH concentrations in sea lions from the no DA exposure group but not in sea lions in the acute or chronic DA toxicosis groups. Following cosyntropin injection, fecal glucocorticoid concentrations increased in all sea lions evaluated except 1. In adult sea lions, eosinophilia may be a cost-effective biomarker for DA exposure and may reflect alterations in hypothalamic, pituitary gland, or adrenal gland function. Domoic acid exposure may have subtle health effects on marine animals in addition to induction of neurologic signs.

Magnetic resonance imaging reveals that brain atrophy is more severe in older California sea lions with domoic acid toxicosis

In 1998, domoic acid (DA) toxicosis was first documented in marine mammals, when more than 400 California sea lions (Zalophus californianus) were determined to have been exposed to DA through contaminated prey that was linked to a bloom of toxin-producing diatoms. Over the last fifteen years, these blooms have increased in frequency and distribution, and DA toxicosis has become a more complex disease. Evidence with laboratory animals show that DA can cause epilepsy, may affect brain development, and may have synergistic effects with some pollutants. Detecting these effects in marine mammals requires quantitative methods to evaluate brain morphology in live animals. In this study, our goal was to employ magnetic resonance imaging (MRI) to examine the morphologic effects of DA toxicosis on the brain in live, wild California sea lions admitted to a rehabilitation center. We performed MRIs and volumetrics of brain structures of 53 sea lions that exhibited clinical signs of acute or chronic DA intoxication. We found that the volumes of the hippocampi and parahippocampal gyri of sea lions with chronic DA toxicosis were less than the volumes of these structures in non-DA intoxicated animals. In addition, adult sea lions with chronic DA toxicosis had more structural damage to the brain than younger animals. This pattern may be explained by one or more of the following possibilities: (i) repetitive, sublethal exposure; (ii) increased susceptibility of adults to DA compared to younger animals; and/or (iii) initial exposure to the toxin followed by the progressive effects of ongoing seizure activity. Of these three possibilities, increased susceptibility and progressive effects of ongoing seizure activity most likely explain why atrophy of the hippocampus and thinning of the parahippocampal gyrus is most severe in adults.

A novel antibody-based biomarker for chronic algal toxin exposure and sub-acute neurotoxicity.

The neurotoxic amino acid, domoic acid (DA), is naturally produced by marine phytoplankton and presents a significant threat to the health of marine mammals, seabirds and humans via transfer of the toxin through the foodweb. In humans, acute exposure causes a neurotoxic illness known as amnesic shellfish poisoning characterized by seizures, memory loss, coma and death. Regular monitoring for high DA levels in edible shellfish tissues has been effective in protecting human consumers from acute DA exposure. However, chronic low-level DA exposure remains a concern, particularly in coastal and tribal communities that subsistence harvest shellfish known to contain low levels of the toxin. Domoic acid exposure via consumption of planktivorous fish also has a profound health impact on California sea lions (Zalophus californianus) affecting hundreds of animals yearly. Due to increasing algal toxin exposure threats globally, there is a critical need for reliable diagnostic tests for assessing chronic DA exposure in humans and wildlife. Here we report the discovery of a novel DA-specific antibody response that is a signature of chronic low-level exposure identified initially in a zebrafish exposure model and confirmed in naturally exposed wild sea lions. Additionally, we found that chronic exposure in zebrafish caused increased neurologic sensitivity to DA, revealing that repetitive exposure to DA well below the threshold for acute behavioral toxicity has underlying neurotoxic consequences. The discovery that chronic exposure to low levels of a small, water-soluble single amino acid triggers a detectable antibody response is surprising and has profound implications for the development of diagnostic tests for exposure to other pervasive environmental toxins.

Pseudo‐nitzschia blooms, domoic acid, and related California sea lion strandings in Monterey Bay, California

AbstractBlooms of the toxin‐producing diatom Pseudo‐nitzschia commonly occur in Monterey Bay, California, resulting in sea lion mortality events. The links between strandings of California sea lions suffering from domoic acid (DA) toxicity, toxic cell numbers, and their associated DA concentration in Monterey Bay and in sea lion feces were examined from 2004 to 2007. While Pseudo‐nitzschia toxic cells and DA concentrations were detectable in the water column most of the time, they were often at low levels. A total of 82 California sea lions were found stranded in the Bay between 2004 and 2007 with acute or chronic signs associated with DA poisoning. The highest number with detectable DA in feces occurred in April 2007 and corresponded with the presence of a highly toxic bloom in the Bay. Higher DA levels occurred in feces from sea lions stranding with acute toxicosis and lower concentrations in feces of sea lions exhibiting signs of chronic DA poisoning or not exhibiting any neurologic signs. Results indicated that sea lions are likely exposed to varying levels of DA through their prey throughout the year, often at sublethal doses that may contribute to a continued increase in the development of chronic neurologic sequelae.