Leander Tryphonas

Acute Neurotoxicity of Domoic Acid in the Rat

A recent outbreak of human food poisoning, characterized by severe gastrointestinal and neurologic abnormalities, with a fatal outcome in 3 patients, was attributed to the consumption of poisonous mussels containing domoic acid at an abnormally high concentration. The purpose of the present study was to determine if domoic acid, a glutamate analogue extracted from poisonous mussel, was neurotoxic to rats. Groups of female Sprague-Dawley rats were dosed once intraperitoneally with 0, 1, 2, 4, or 7.5 mg domoic acid/kg of body weight and observed for a maximum period of 24 hr. Clinically, control rats and rats in the 1 mg/kg group were unremarkable. Seventy-five percent of the animals in the 2 mg/kg group had equivocal transient behavioral signs. One that was given 2 mg/kg and all rats given in excess of 4 mg/kg of body weight developed unequivocal behavioral and neurologic signs culminating in partial seizures and status epilepticus. Histopathologically, severely affected rats developed selective encephalopathy characterized by neuronal degeneration and vacuolation of the neuropil in the limbic and the olfactory systems, and retinopathy characterized by neuronal hydropic degeneration of the inner nuclear layer and vacuolation of the external plexiform layer. The results of this study suggest that domoic acid is excitotoxic and causes a characteristic syndrome with clinical signs and histopathologic lesions similar to those reported for kainic acid.

Domoic acid poisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract.

Consumption of cultivated blue mussels from Prince Edward Island was recently associated with episodes of gastro-intestinal and neurological distress. Extracts of the toxic mussels, tested in the mouse bioassay for paralytic shellfish poison, caused an atypical response characterized by scratching, convulsions and death. The present investigation shows that the domoic acid present in toxic mussels can produce in mice and rats signs identical to those induced by mussel extracts. These studies, preliminary in nature by virtue of the scarcity of domoic acid, gave ip no-effect levels in mice of 0.59 mg/kg body weight based on the behavioural response (scratching) and 2.4 mg/kg for death. These levels correspond to levels of 24 and 94 ppm in mussels. When administered orally doses of between 35 and 70 mg domoic acid/kg body weight were required to produce toxicity in mice and rats. This reduced toxicity is consistent with a lack of absorption from the gastro-intestinal tract: faecal excretion accounted for 102 +/- 17% and 98 +/- 12% (mean +/- SE) of the domoic acid administered to mice and rats, respectively. Since human intoxication occurred at an estimated 1-5 mg domoic acid/kg body weight, susceptible individuals appear to be more sensitive than rodents to the oral toxicity of domoic acid.

Acute Parenteral Neurotoxicity of Domoic Acid in Cynomolgus Monkeys (M. fascicularis)

To study the CNS effects of domoic acid (D.A.), 6 adult Cynomolgus monkeys (M. fascicularis) were dosed intraperitoneally (4 mg/kg) or intravenously (0.025-0.5 mg/kg) with D.A. obtained from cultured mussels contaminated with this neurotoxin. Clinical signs of neurotoxicity were preceded by a short presymptomatic period (2-3 min) and an even shorter prodromal period (0.5-1 min). The symptomatic period proper was characterized by persistent chewing with frothing, varying degrees of gagging, and vomit. Monkeys in the higher dose regimen exhibited additional signs including abnormal head and body positions, rigidity of movements and loss of balance, and tremors. The duration of the symptomatic period was dose dependent. Excitotoxic lesions consisting of vacuolation of the neuropil, astrocytic swelling, and neuronal shrinkage and hyperchromasia were detected in the area postrema, the hypothalamus, the hippocampus, and the inner layers of the retina in monkeys given D.A. at 0.5 mg/kg intravenously and 4 mg/kg intraperitoneally. It was concluded that D.A., administered intravenously, is neuroexcitatory and a powerful emetic at doses of 0.025 to 0.2 mg/kg. At higher doses (0.5 mg/kg intravenously and 4 mg/kg intraperitoneally), D.A. is strongly excitotoxic.

Neuropathology of excitatory neurotoxins: the domoic acid model.

A novel type of intoxication in Canada in 1987 was traced to consumption of cultivated mussels contaminated with the excitotoxin domoic acid. Studies carried out in rats and monkeys revealed that parenterally administered domoic acid induces in rats neuroexcitatory phenomena culminating in seizures. Monkeys respond with gagging, emesis and less clearly evident seizure activity. CNS damage consisting of dendrotoxic and gliotoxic edema and nerve cell degeneration occurs in structures of the limbic system and the retina in both species. CNS lesion distribution similarities in animals treated with domoic acid or kainic acid suggest that these excitotoxins share a common pathogenesis mediated by glutamic acid, a putative endogenous excitatory neurotransmitter.

Experimental oral toxicity of domoic acid in cynomolgus monkeys (Macaca fascicularis) and rats.: Preliminary investigations☆

A recent outbreak of marine food poisoning in humans was attributed to the consumption of blue mussels (Mytilus edulis L.) contaminated with domoic acid (DA) that was produced by the diatom Nitzschia pungens. The clinical and morphological effects of single oral doses of extracts of mussels contaminated with DA or of DA isolated from toxic mussels were investigated in small groups (one to six) of cynomolgus monkeys (Macaca fascicularis; 0.5-10 mg DA/kg body weight) and of Sprague-Dawley rats (60 to 80 mg DA/kg body weight). Control animals were either given saline or were not treated. To test whether monosodium glutamate, present in the food consumed by some affected humans, and dimethylsulphoxide, suspected of being present in the plankton, enhanced the response, monosodium glutamate (at 0.25% of mussel extract bolus) or dimethylsulphoxide (at 1 g per bolus) were co-administered to two (one each) of the DA-treated monkeys. DA-treated monkeys developed transient excitation characterized by vomiting. DA-treated rats showed withdrawal followed by hyperexcitation and death (in one case). Mild to moderate central nervous system lesions consistent with neuroexcitation were present in both monkeys and rats. The addition of monosodium glutamate and dimethylsulphoxide had no significant effect on the appearance and severity of central nervous system clinical signs and lesions. The wide variations in the response of test animals to orally administered DA were attributed to the protective effect of vomiting, and to suspected incomplete or slow gastro-intestinal absorption of the toxic agent. The results reinforce the view that DA is an emetic and that under appropriate conditions may also inflict excitotoxic central nervous system damage.

Polychlorinated biphenyl toxicity in the pregnant cynomolgus monkey: A pilot study

Three pregnant cynomolgus monkeys (Macaca fascicularis) were dosed with 100 or 400 μg/kg/day of Aroclor® 1254 from approximately 60 days of gestation. One additional pregnant monkey was given dose vehicle only. The two monkeys dosed with 100 μg/kg/day delivered stillborn infants and the 400 μg/kg/day dosed monkey delivered a term infant that had impaired immunologic function compared with the control infant, and died at 139 days post partum. The three dams also had impaired immunologic capacity assessed at approximately 50 days post partum (148 days treatment). With the exception of loss of fingernails in two monkeys, no overt clinical signs of toxicity were observed in the adults. Polychlorinated biphenyl (PCB) concentrations and peak ratios in breast milk and tissues are reported.

Polychlorinated biphenyl (PCB) Toxicity in Adult Cynomolgus Monkeys (M. fascicularis): A Pilot Study

Aroclor 1254 and Aroclor 1248, at doses of 11.7 and 4.7 mg/kg body weight (equivalent to 5 and 2 mg/kg/day), were given 3 days per week to groups of cynomolgus monkeys, and caused weight loss, fingernail loss, facial edema, epiphora, and death. Blood and adipose tissue PCB concentrations rose with the length of treatment. Tissue concentrations in blood, adipose tissue, liver and kidneys were highest in monkeys treated with Aroclor 1254, reflecting dose differences. There was considerable variation, both within and between groups, in hematologic responses to PCB treatment. Aroclor 1254-treated monkeys had depressed and weakly responsive erythropoiesis. Aroclor 1248-treated monkeys had active but ineffective or depressed erythropoiesis with severe macrocytic or moderate normocytic anemia. Biochemical determination of blood serum constituents revealed treatment and time-related trends towards hypoalbuminemia and increased alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, lactic dehydrogenase, cholesterol, triglycerides, total bilirubin and direct bilirubin values. Pathologic lesions common in both Aroclor groups were dilatation of meibomian glands duct; mucinous hyperplasia of the gastric mucosa; atrophy and loss of germinal centers in the splenic and other lymphoid follicles; enlargement, fatty degeneration, and necrosis of hepatocytes; bile duct and gall bladder epithelial cell hypertrophy and hyperplasia; and thyroid aberrations in follicular cell size and number of intracytoplasmic lysosomes. Lesions seen exclusively in an Aroclor 1254-treated monkey were widespread mucinous metaplasia and hyperplasia of the fundic mucosa. The results suggest that in general, cynomolgus monkeys may be more refractory or less susceptible to PCB toxicity than rhesus monkeys and, that Aroclor 1248 may be more toxic than Aroclor 1254.

Histomorphogenesis of Soman-Induced Encephalocardiomyopathy in Sprague-Dawley Rats

Although myocardial damage caused by soman has been previously reported, its relation to brain damage is unclear. In order to clarify this relationship, we examined the histomorphogenesis of central nervous system (CNS) and myocardial lesions in Sprague-Dawley rats, given atropine methylnitrate (20 mg/kg) and HI-6 (125 mg/kg) ip 10 min before a single injection of 0 or 130 μg soman/kg (sc) and sacrificed 45 min and 1.5 hr, 3 hr, 24 hr, and 72 hr later. Bilaterally symmetrical CNS damage began with vacuolation of the neuropil and was followed by astrocytic degeneration and neuronal necrosis culminating in liquefaction necrosis and focal hemorrhage. The cerebral cortex, limbic system, thalamus, and substantia nigra were common target sites. Repair in affected sites was characterized by capillary endothelial cell proliferation, microgliosis, and reversal of microvacuolation. Myocardial damage began with myocytolysis and contraction bands and evolved into coagulative myocytolysis and replacement fibrosis with a transient recruitment of acute inflammatory cells. The left ventricle, especially its free wall and papillary muscles, was consistently affected. There was good correlation among seizures, CNS damage, and myocardial lesions at all times following treatment. The results support the view that CNS lesions are associated with protracted seizure activity and provide evidence that myocardial damage is neurogenic.

A pilot study in adult rhesus monkeys (M. mulatta) treated with Aroclor 1254 for two years.

Aroclor 1254, at a dose level of 280 μg/kg body weight equivalent to 200 μg/kg/day, was given 5 days per week to rhesus monkeys over a 27 to 28 month period. Terminal clinical signs of varying severity included fingernail detachment, exuberant nail beds, weight loss, stomatitis and normocytic anemia. At necropsy the bone marrow was hypocellular with increased M:E ratio and cytoplasmic vacuoles in crythroid precursor cells. Histopathologic lesions included dilatation of the tarsal gland ducts, atrophy or absence of splenic and lymphonodal germinal centers, bone marrow depletion, gingival erosion and ulceration, moderate mucinous hypertrophic gastropathy with cystic dilatation of occasional gastric glands, hepatocellular enlargement and necrosis, hypertrophy of biliary duct epithelium, hyperplasia of biliary ducts, hypertrophy of the gall bladder epithelium, and an equivocal increase in the number of lysosomes in thyroid follicular epithelial cells. PCB tissue concentrations were lowest in brain and highest in blood. The results suggest that severe potentially fatal PCB toxicity can develop in rhesus monkeys following ingestion of Aroclor 1254 at 200 μg/kg/day for a period of 27 months or longer.

Comparative aspects of Aroclor® 1254 toxicity in adult cynomolgus and rhesus monkeys: A pilot study

Aroclor® 1254 (PCB), at a dose of 280 μg/kg body weight (equivalent to 200 μg/kg/day) was given 5 days per week to groups of cynomolgus and rhesus monkeys. Cynomolgus monkeys were treated for 12 to 13 months and rhesus monkeys for 27 to 28 months. The study compares selected clinical, hematologic, immunologic, and analytical findings in the two species up to the time the cynomolgus were killed (12–13 months) and includes the pathologic findings in the latter (terminal findings in the rhesus are the subject of a separate report). Treated rhesus had enlarged tarsal glands, conjunctivitis, loss of eyelashes, progressive detachment of finger nails, exuberant nail beds, and somewhat depressed hematologie values of the erythron. In contrast, treated cynomolgus had temporary enlargement of the tarsal glands, distortion and temporary loss or lifting of nails with limited exposure of the nail beds, and moderate erythroid depression and vacuolization of early erythroid precursors. Histopathologic findings in the cynomolgus monkeys included mild dilatation of tarsal gland ducts, moderately keratinized finger nail beds, and hepatocellular and biliary duct epithelial cell hypertrophy. Immunologie testing was inconclusive due to large interspecies variability. PCB analysis revealed that during the first 18 weeks of treatment rhesus monkeys accumulated PCB faster than did the cynomolgus. It appears that rhesus monkeys are more susceptible to PCB toxicity than cynomolgus monkeys.