Prolonged, Low-Level Exposure to the Marine Toxin, Domoic Acid, and Measures of Neurotoxicity in Nonhuman Primates

Background: The excitotoxic molecule, domoic acid (DA), is a marine algal toxin known to induce overt hippocampal neurotoxicity. Recent experimental and epidemiological studies suggest adverse neurological effects at exposure levels near the current regulatory limit (20 ppm, ∼0.075–0.1mg/kg). At these levels, cognitive effects occur in the absence of acute symptoms or evidence of neuronal death. Objectives: This study aimed to identify adverse effects on the nervous system from prolonged, dietary DA exposure in adult, female Macaca fascicularis monkeys. Methods: Monkeys were orally exposed to 0, 0.075, and 0.15mg/kg per day for an average of 14 months. Clinical blood counts, chemistry, and cytokine levels were analyzed in the blood. In-life magnetic resonance (MR) imaging assessed volumetric and tractography differences in and between the hippocampus and thalamus. Histology of neurons and glia in the fornix, fimbria, internal capsule, thalamus, and hippocampus was evaluated. Hippocampal RNA sequencing was used to identify differentially expressed genes. Enrichment of gene networks for neuronal health, excitotoxicity, inflammation/glia, and myelin were assessed with Gene Set Enrichment Analysis. Results: Clinical blood counts, chemistry, and cytokine levels were not altered with DA exposure in nonhuman primates. Transcriptome analysis of the hippocampus yielded 748 differentially expressed genes (fold change≥1.5; p≤0.05), reflecting differences in a broad molecular profile of intermediate early genes (e.g., FOS, EGR) and genes related to myelin networks in DA animals. Between exposed and control animals, MR imaging showed comparable connectivity of the hippocampus and thalamus and histology showed no evidence of hypomyelination. Histological examination of the thalamus showed a larger microglia soma size and an extension of cell processes, but suggestions of a GFAP+astrocyte response showed no indication of astrocyte hypertrophy. Discussion: In the absence of overt hippocampal excitotoxicity, chronic exposure of Macaca fascicularis monkeys to environmentally relevant levels of DA suggested a subtle shift in the molecular profile of the hippocampus and the microglia phenotype in the thalamus that was possibly reflective of an adaptive response due to prolonged DA exposure. https://doi.org/10.1289/EHP10923


Table of Contents
Serum Chemistry Reactions on the Beckman Coulter AU System. Table S1. Animal Characteristics. Table S2. Curated Gene Lists from Literature, Used in GSEA. Figure S1. Matching H&E staining for focal sites of microglia reactivity. Representative images of H&E staining in 10% formalin-fixed, paraffin-embedded, 10 µm sections at focal sites of reactivity in the thalamus, fornix, fimbria, internal capsule, and nucleus accumbens of female Macaca fascicularis following prolonged exposed to domoic acid (0.15 mg/kg/d) or vehicle (5% sucrose). Numbers correspond to Animal Numbers in Table S1. A15244 and A16106 were in the 0.15 mg/kg/d group, A15249, A16107, and A16106 were in the 0.075 mg/kg/d group, and A15428 was in the control group. Scale bar = 60 µm.
Additional File-Excel Document 1. Sodium, Potassium, and Chloride The Beckman Coulter AU System ISE module for Na+, K+, and Cl-employs crown ether membrane electrodes for sodium and potassium; and a molecular oriented PVC membrane for chloride that are specific for each ion of interest in the sample. An electrical potential was developed according to the Nernst Equation for a specific ion. When compared to the Internal Reference Solution, this electrical potential is translated into voltage and then into the ion concentration of the sample.

Urea Nitrogen
Urea was hydrolyzed enzymatically by urease to yield ammonia and carbon dioxide. The ammonia and α-oxoglutarate were converted to glutamate in a reaction catalyzed by L-glutamate dehydrogenase (GLDH). Simultaneously, a molar equivalent of reduced NADH was oxidized. Two molecules of NADH were oxidized for each molecule of urea hydrolyzed. The rate of change in absorbance at 340 nm, due to the disappearance of NADH, is directly proportional to the BUN concentration in the sample.

Total Protein
Cupric ions in an alkaline solution reacted with proteins and polypeptides containing at least two peptide bonds to produce a violet-colored complex. The absorbance of the complex at 540/660 nm is directly proportional to the concentration of protein in the sample.

Albumin
At pH 4.2, bromocresol green reacts with albumin to form an intense green complex. The absorbance of the albumin-BCG complex was measured bichromatically (600/800nm), proportional to the albumin concentration in the sample.

Total Bilirubin
Total bilirubin in serum is composed of direct (conjugated) bilirubin and indirect (unconjugated) bilirubin. A stabilized diazonium salt, 3,5-dichlorophenyl-diazonium tetrafluoroborate (DPD), reacted with bilirubin to form azobilirubin, which absorbs maximally at 570/660 nm. Caffeine and a surfactant were used as reaction accelerators. The absorbance at 570/660 nm is proportional to the bilirubin concentration in the sample. A separate serum blank was performed to eliminate endogenous serum interferences.

Calcium
Calcium ions (Ca2+) react with Arsenazo III (2,2'-[1,8-Dihydroxy-3,6-disulphonaphthylene-2,7-bisazo]bisbenzenear-sonic acid) to form an intense purple colored complex. Magnesium does not significantly interfere in calcium determination using Arsenazo III. In this method, the absorbance of the Ca-Arsenazo III complex was measured bichromatically at 660/700 nm. The resulting increase in absorbance of the reaction mixture is directly proportional to the calcium concentration in the sample.

Phosphate
Inorganic phosphate reacts with molybdate to form a heteropolyacid complex. The use of a surfactant eliminates the need to prepare a protein free filtrate. The absorbance at 340/380 nm was measured in this reaction as directly proportional to the inorganic phosphorus level in the sample.

Cholesterol
Cholesterol esters in serum are hydrolyzed by cholesterol esterase (CHE), using reagents certified to meet the National Cholesterol Education Program's (NCEP) performance criteria for accuracy. The free cholesterol produced was oxidized by cholesterol oxidase (CHO) to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide (H202), which oxidatively couples with 4aminoantipyrine and phenol in the presence of peroxidase to yield a chromophore. The red quinoneimine dye formed was measured spectrophotometrically at 540/600 nm as an increase in absorbance.
9. Alkaline Phosphatase Alkaline phosphatase activity was determined by measuring the rate of conversion of p-nitrophenylphosphate (pNPP) in the presence of 2-amino-2-methyl-1-propanol (AMP) at pH 10.4. The rate of change in absorbance due to the formation of pNP was measured bichromatically at 410/480 nm and is directly proportional to the ALP activity in the sample.

Alanine Aminotransferase
This method utilizes a methodology recommended by the International Federation of Clinical Chemistry (IFCC). ALT transfers the amino group from alanine to α-oxoglutarate to form pyruvate and glutamate. The pyruvate enters a lactate dehydrogenase (LD) catalyzed reaction with NADH to produce lactate and NAD+. The decrease in absorbance due to the consumption of NADH was measured at 340nm and is proportional to the ALT activity in the sample.

Aspartate Aminotransferase
This method uses methodology recommended by the IFCC. In this method, aspartate aminotransferase (AST) catalyzes the transamination of aspartate and α-oxoglutarate, forming L-glutamate and oxalacetate. The oxalacetate is then reduced to L-malate by malate dehydrogenase, while NADH is simultaneously converted to NAD+. The decrease in absorbance due to the consumption of NADH was measured at 340 nm and is proportional to the AST activity in the sample.