Subchronic neurotoxicity screening studies with six organophosphate insecticides: an assessment of behavior and morphology relative to cholinesterase inhibition.

Sulprofos, disulfoton, azinphos-methyl, methamidophos, trichlorfon, and tebupirimphos were screened for neurotoxic potential, in accordance with U.S. EPA (FIFRA) requirements. Each organophosphate was administered through the diet for 13 weeks to separate groups of Fischer 344 rats at four dose levels, including a vehicle control. For each study, 12 rats/sex/dietary level were tested using a functional observational battery (FOB), automated measures of activity (figure-8 maze), and detailed clinical observations, with half of the animals perfused at term for microscopic examination of neural and muscle tissues. Separate groups of satellite animals (6/sex/dietary level) were used to measure the effect of each treatment on plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity. The results show that measures of ChE activity were consistently the most sensitive indices of exposure and assisted in the interpretation of findings. All treatment-related neurobehavioral findings were ascribed to cholinergic toxicity, occurring only at dietary levels that produced more than 20% inhibition of plasma, RBC, and brain ChE activity. Neurobehavioral tests provided no evidence of additional cumulative toxicity after 8 weeks of treatment. The FOB and motor activity findings did not alter the conclusions and generally did not reduce the neurobehavioral no-observed-effect level (NOEL) for any of the six compounds, relative to the results from detailed clinical observations as conducted in these studies. The one exception occurred with tebupirimphos, where the NOEL for motor activity was one dose level lower than the NOEL for the FOB and clinical observations. These results support the value of detailed clinical observations to screen for the neurotoxic potential of organophosphates and a general standard of more than 20% inhibition of brain ChE activity for cholinergic neurotoxicity.

Associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles.

The thyroids were evaluated in 276 control Beagles that were allowed to live out their full life span (mean = 12 years) in a closed breeding colony. Lymphocytic thyroiditis was found in 26.3% of the dogs. This lesion was characterized by lymphoplasmacytic inflammation accompanied by follicular destruction. The thyroiditis was progressive, resulting in severe atrophy of follicular tissue, and 44 dogs (15.9%) were diagnosed as hypothyroid at the time of death. In accordance with the experimental protocol, hypothyroid dogs were not given thyroxine replacement therapy. There was a high degree of heritability for the hypothyroidism. Hypothyroid dogs had an increased risk for thyroid follicular epithelial neoplasia and, in particular, for follicular adenocarcinomas. Twenty-four of the 44 hypothyroid dogs (54.5%) had one or more follicular thyroid neoplasms, whereas only 53 of the 232 (22.8%) clinically euthyroid dogs had similar tumors. Multiple thyroid tumors were present in 14 of the 44 (31.8%) hypothyroid dogs but in only 12 of the 232 (5.2%) euthyroid dogs. One or more follicular adenocarcinomas were present in 15 of the 44 (34.1%) hypothyroid dogs but in only 16 of the 232 (6.9%) euthyroid dogs. There was no difference in prevalence of hypothyroidism or tumors between the sexes. The strong association between progressive lymphocytic thyroiditis, hypothyroidism, and thyroid follicular neoplasia in these Beagles probably relates to promotion of residual follicular epithelium by chronic excess thyrotropin stimulation.

The effects of physostigmine on the electroretinogram in the beagle dog.

The purpose of the study was to correlate electroretinogram (ERG) parameters with increasing levels of plasma, erythrocyte and ocular tissue cholinesterase inhibition using the beagle dog as a model for human neurovisual toxicity. The anticholinesterase compound physostigmine was administered at various steady-state intravenous infusion rates based on pharmacokinetic estimates of plasma and red blood cell cholinesterase inhibition. The most sensitive parameter was the b-wave amplitude of the rod response, which was significantly depressed compared to pretreatment at all levels of acute cholinesterase depression. The overall maximal ERG response demonstrated a trend of declining a- and b-wave amplitudes, which corresponded with the increased levels of cholinesterase depression, but these differences were not significant. The depression of the electroretinogram rod and cone amplitudes appeared to parallel plasma cholinesterase inhibition more closely than erythrocyte cholinesterase activity. Ocular tissue cholinesterase activity was significantly depressed in the retina (70%), cornea (60%) and dorsal rectus extraocular muscle (46%). Electroretinography may be a useful physiological tool for evaluating the ocular toxicity of certain chemicals or pharmaceuticals associated with cholinesterase biomarker activity.

The effect of perinatal 60Co gamma radiation on brain weight in beagles.

Beagle dogs were given single, whole-body 60Co gamma-radiation exposures at one of three prenatal (8, 28, or 55 days postcoitus) or three postnatal (2, 70, or 365 days postpartum) ages to evaluate the relative radiosensitivity of various stages of brain development. A total of 387 dogs received mean doses ranging from 0.16 to 3.83 Gy, and 120 dogs were sham-irradiated. Groups of dogs were sacrificed at preselected times from 70 days to 11 years of age. Brain weight decreased significantly with increasing dose in dogs irradiated at 28 or 55 days postcoitus or at 2 days postpartum. Irradiations at 28 days postcoitus were dramatically more effective in causing a reduction in brain weight than those at 55 days postcoitus or 2 days postpartum. Among dogs given 1.0 Gy or more and followed for up to 4 years, there was a radiation effect evident at all three sensitive exposure ages. Among dogs given lower doses and followed for up to 11 years, there was a significant decrease in brain weight in dogs given 0.80-0.88 Gy at 28 days postcoitus. All decreases in brain weight were present after normalization for radiation-induced reductions in skeletal (body) size. No specific morphologic changes were noted in the brains which showed the radiation-related reductions in size.

Trabecular bone morphometry in beagles with hyperadrenocorticism and adrenal adenomas.

Trabecular bone morphometry was done on rib samples of beagles with hyperadrenocorticism and adrenal adenomas to evaluate bone loss and the remodeling changes responsible. Beagles diagnosed as having clinical hyperadrenocorticism and those with milder or subclinical hyperadrenocorticism diagnosed on the basis of adrenal and pituitary lesions at necropsy had increased adrenal and pituitary gland weights. In a group of dogs with adrenal cortical adenomas there was atrophy of remaining cortex, and the combined weight of adrenal glands or pituitary weights were not increased. In dogs with clinical hyperadrenocorticism, mean trabecular bone volume was 25% less than controls (P = 0.10). In both clinical and subclinical hyperadrenocorticism groups, the extent of trabecular surface with unmineralized osteoid matrix and osteoblasts was significantly reduced. There were no changes in resorption surfaces or number of osteoclasts present. No bone changes were seen in dogs with adrenal adenomas. In dogs with hyperadrenocorticism it appeared that decreased bone formation was primarily responsible for the relative osteopenia that developed. Although parathyroid glands were moderately enlarged in those dogs for which weights were available, the bone changes were not those of increased remodeling expected in hyperparathyroidism.

Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage.

The clinical signs and morphological brain lesions associated with histotoxic hypoxia induced by subcutaneous injection of 3-nitropropionic acid (NPA) in rats are described, and compared to hypoxic brain damage from other causes including ischemia and hypoglycemia. The brains were perfusion-fixed with paraformaldehyde/glutaraldehyde fixative, and examined by light and electron microscopy. Intoxicated rats developed severe neurological disease characterized by somnolence, uncoordinated gait with stereotypical paddling movements, and ventral or lateral recumbency. Recumbent rats had a selective, bilaterally symmetrical pattern of severe morphological injury in the caudate-putamen, hippocampus, and thalamus. Recumbency was a consistent indicator of the development of morphological brain lesions. In contrast to reports describing rat models of ischemia and hypoglycemia, morphological injury was not seen in the cerebral and cerebellar cortices of NPA-intoxicated rats. Ultrastructurally, neuronal alterations ranged from chromatin clumping with increased cytoplasmic lucency to severe cellular shrinkage or swelling with marked mitochondrial swelling (high amplitude swelling). White matter alterations included axonal swelling and adaxonal splitting of myelin lamellae. Vascular changes included perivascular deposits of proteinaceous material presumably from leakage of serum proteins, variable electron lucency of endothelial cell cytoplasm, an apparent increase in pinocytotic vesicles, rare platelet thrombosis of capillaries, and rare intravascular blebs of luminal plasma membrane. As a model of brain damage following energy deficiency, NPA intoxication has the advantages of producing morphological brain injury in a highly predictable anatomical pattern, and at a time paralleling the onset of clinical recumbency.

Correlation of morphologic brain lesions with physiologic alterations and blood-brain barrier impairment in 3-nitropropionic acid toxicity in rats.

3-Nitropropionic acid (NPA), a toxin which irreversibly inhibits the Krebs cycle enzyme succinate dehydrogenase, causes severe neurologic disease and a specific pattern of morphologic brain damage when given subcutaneously to rats. To determine whether hypotension or hypoxemia were necessary for development of morphologic brain lesions in NPA neurotoxicity, systemic blood pressure and arterial blood gases were measured in NPA-intoxicated rats. The extent and distribution of albumin extravasation was examined by immunohistochemistry, and was compared to the extent and severity of morphological injury in the caudate-putamen. Neither hypotension nor hypoxemia were necessary for the development of morphologic injury in the brains of NPA-intoxicated rats. In fact, intoxicated rats had significantly higher systolic blood pressure and arterial blood oxygen than did controls. Arterial bicarbonate and pH were significantly lower in intoxicated rats than controls, however, suggesting that acidosis may be involved in the pathogenesis of NPA toxicity. When morphologic injury was severe, albumin extravasation was extensive occupying approximately 30%-80% of the lesion area in the caudate-putamen of NPA-intoxicated rats. When morphologic injury was mild, albumin extravasation was absent, or limited to small cuffs around individual capillaries (less than 1% of the lesion area). There was no leakage of albumin in the cerebral cortex, which was resistant to morphologic injury. It was concluded that leakage of protein-rich fluid into cerebral parenchyma from blood-brain barrier impairment is not responsible for the initiation of morphologic injury in NPA toxicity, but may contribute to the severity of injury later in the evolution of brain lesions.