The Autosow raised miniature swine as a model for assessing the effects of dietary soy trypsin inhibitor.

Toxicological effects of dietary soy trypsin inhibitor (TI) were assessed in male miniature swine, a model chosen for its similarities to human digestive physiology and anatomy. The TI preparation was extracted from defatted raw soy flour. From 1 through 5 weeks of age, piglets were automatically fed either a TI liquid diet [Autosow TI group (ASTI)] or a control liquid diet [Autosow control group (ASC)]. From 6 to 39 weeks of age, these animals received either swine chow and TI or swine chow and control article. The TI diets were formulated to contain a TI activity of approximately 500 mg TI/100 g dry matter. A sow control (SC) group suckled from birth to 6 weeks of age and then fed as the ASC group with swine chow plus control article from 6 to 39 weeks of age. The SC piglets grew faster than ASC piglets during postnatal weeks 1 and 2; however, the ASC piglets were significantly heavier than the SC piglets (P=0.001) at 6 weeks of age. Compared with the ASC group, TI caused a moderate decrease in feed consumption and a moderate but reversible decrease in growth from 2 to 5 weeks of age, but not thereafter. Some control and TI-fed Autosow-reared piglets had loose stools until 6 weeks of age; the effect was significantly greater in the TI-fed group. Otherwise, all swine were active and had normal appearance and behavior.

Pathological evaluation, clinical chemistry and plasma cholecystokinin in neonatal and young miniature swine fed soy trypsin inhibitor from 1 to 39 weeks of age.

The potential toxicity of dietary soy trypsin inhibitor (TI) was evaluated in neonatal miniature swine. From 1 to 6 weeks of age, two groups of male piglets were artificially reared in an Autosow and automatically fed either TI or control liquid diet. From 6 to 39 weeks of age, these two groups were fed either TI or control chow diet. A third group, sow control (SC), suckled from birth to 6 weeks of age, were also weaned to control chow from 6 to 39 weeks of age. Clinical chemistry and plasma cholecystokinin (CCK) determined at 6, 18, 30 and 39 weeks of age, and serum amylase activity with gross and histopathological analyses of major organs at 6 and 39 weeks of age are reported. TI had no effect on plasma CCK, serum amylase activity, or numerous clinical chemistry values. TI-fed piglets had a larger relative liver weight at 6 weeks of age. Relative pancreas weight decreased with age but was not affected by TI. Gross and histopathological analyses of major organs, except the spleen, were within normal limits. Increased incidence of extramedullary hematopoiesis was noted in the spleen of the TI group at 6 but not at 39 weeks of age. There was no consistent pattern in immunohistochemical foci for secretin, gastrin releasing polypeptide or CCK, and no change in DNA, RNA, mitotic index or nuclear density of pancreatic cells. At 6 weeks of age, TI increased pancreatic protein and amylase activity but not trypsin or chymotrypsin activity. None of the effects suggested that this dose of TI was toxic to either the neonatal or sexually mature miniature male swine.

From farm to table to brain: foodborne pathogen infection and the potential role of the neuro-immune-endocrine system in neurotoxic sequelae.

The American diet is among the safest in the world; however, diseases transmitted by foodborne pathogens (FBPs) still pose a public health hazard. FBPs are the second most frequent cause of all infectious illnesses in the United States. Numerous anecdotal and clinical reports have demonstrated that central nervous system inflammation, infection, and adverse neurological effects occur as complications of foodborne gastroenteritis. Only a few well-controlled clinical or experimental studies, however, have investigated the neuropathogenesis. The full nature and extent of neurological involvement in foodborne illness is therefore unclear. To our knowledge, this review and commentary is the first effort to comprehensively discuss the issue of FBP induced neurotoxicity. We suggest that much of this information supports the role of a theoretical model, the neuro-immune-endocrine system, in organizing and helping to explain the complex pathogenesis of FBP neurotoxicity.

Persistent neonatal Borna disease virus (BDV) infection of the brain causes chronic emotional abnormalities in adult rats.

Neonatal Borna disease virus (BDV) brain infection results in selective developmental damage to the hippocampal dentate gyrus and the cerebellum. When mature, neonatally BDV-infected rats show extreme locomotor hyperactivity and reduced freezing behavior in novel environments. Traditional interpretation of both of these behavioral abnormalities would suggest decreased anxiety in infected rats compared to normal animals. However, it also possible that the locomotor hyperactivity in infected rats reflects higher rather than reduced anxiety, and is the result of increased escape responses to aversive stimuli. The present experiments were undertaken to test a hypothesis about elevated anxiety in neonatally BDV-infected adult Lewis rats by studying their species-specific fear-related responses. Compared to normal subjects, BDV-infected rats exhibited locomotor hyperactivity and elevated defecation in a highly aversive, brightly lit open field. As expected, in a less aversive, dimly lit open field, uninfected controls increased ambulation, whereas infected rats significantly decreased locomotor activity and defecation. Unlike uninfected rats, BDV-infected rats exhibited an attenuated freezing response immediately after loud auditory stimuli. On the contrary, immediate freezing responses following footshock were comparable in the two groups of animals indicating an intact ability to freeze in BDV-infected rats. Despite a decreased baseline startle responsiveness, BDV-infected rats demonstrated increased sensitization of the startle response by preceding footshocks, suggesting a tendency toward elevated escape responses. Compared to normal subjects, BDV-infected rats showed decreased conditional freezing and elevated conditional defecation response in the context previously paired with aversive stimulation indicating sparing of an autonomic component of fear conditioning. The findings indicate that neonatally BDV-infected adult rats are hyperreactive to aversive stimuli, possibly as a result of chronic emotional abnormalities.

Domoic acid: neurobehavioral and neurohistological effects of low-dose exposure in adult rats.

Adult rats treated IP with domoic acid at 0, 0.22, 0.65, or 1.32 mg/kg were tested for passive avoidance (PA), auditory startle (AS), or conditioned avoidance (CAR) behaviors. Clinical signs were observed only at the 1.32 mg/kg dose level. Within 24 h of dosing, rats surviving a dose of 1.32 mg/kg exhibited transient decreased body weight and exaggerated AS responding. Startle latency and habituation, PA, and CAR were not affected. Examination of brains from six rats per group revealed a subset (2/6) of animals receiving 1.32 mg/kg domoic acid with degenerating neurons in the hippocampal CA1/CA3 subregions and gliosis. The decreased body weight and increased startle suggest a hyperreactivity syndrome possibly related to neuronal degeneration in the hippocampus. In a separate experiment, domoic acid at an IP dose of 0.93 mg/kg was found to produce hypomotility in addition to a decrease in body weight. Both effects were reduced by pretreatment with scopolamine (2 mg/kg), but not with caffeine (30 mg/kg), indicating a possible cholinergic involvement in domoate's toxicity.

Food and Drug Administration Proposed Guidelines for Neurotoxicological Testing of Food Chemicals.

The fact that some chemicals may adversely affect the nervous system is certainly not a new concept in regulatory toxicology. In 1982, the FDA issued testing guidelines for the safety evaluation of proposed direct food and color additives which included the assessment of nervous system toxicity as part of the general toxicological profile. However, these guidelines provide only broad and nonspecific recommendations as to how this assessment may best be carried out. The information derived from toxicity screening studies conducted according to these guidelines enable little more than the detection of clearly evident adult nervous system toxicity associated with general neuropathology and overt neurological dysfunction. Little consistent or systematically documented information is typically developed about other equally important types of neurotoxic effects including, for example, behavioral dysfunction and developmental neurotoxicity. Concerns about these more subtle types of neurotoxic effects have become a prominent public health issue and have resulted in demands for an increasing level of assurance that efforts are being made to minimize even further the risks of neurotoxicity from human exposure to chemical substances. In an effort to address these concerns, the FDA is including specific attention to neurotoxicity in a proposed revision of its toxicity testing guidelines for food additives. These proposed guidelines focus on a more careful evaluation of structural and functional measures of neurotoxicity as a routine component of safety assessment. This focus will enable the development of the type of information needed for a more effective assessment of the full spectrum of neurotoxic hazards. The revised guidelines for neurotoxicity testing will be discussed in terms of the FDA's overall approach to safety assessment.

Neurobehavioral dysfunctions associated with dietary iron overload.

Excessive dietary Fe is known to be toxic, but the extent of neurobiological involvement is not clear. In the present study male weanling rats were fed diets containing Fe at 35 (control), 350, 3500, or 20000 ppm for 12 wk. An Fe-deficient group (4 ppm) was included for comparison. Rats were tested for behavioral and body weight changes at various times after initiation of diets, and liver and brain nonheme Fe were measured at term. Excess dietary Fe, primarily at 20000 ppm, significantly decreased activity, habituation, reflex startle, and conditioned avoidance response performance, and enhanced prepulse modulation of startle. Body weights were also markedly decreased. Fe-deficient animals showed similar behavioral effects but more moderate body weight changes. Liver nonheme Fe varied directly with dietary levels. Whole-brain nonheme Fe was significantly reduced in Fe-deficient animals but increased only at the 20000-ppm level. Homeostatic mechanisms appear to regulate whole-brain Fe more effectively under conditions of dietary Fe overload than under conditions of Fe deficiency. The behavioral changes associated with dietary Fe overload may represent secondary consequences of systemic toxicity.

Revisions to the FDA's Redbook guidelines for toxicity testing: neurotoxicity.

In 1982, the Food and Drug Administration issued a publication, known as the Redbook, that described the current toxicological principles used for the safety assessment of regulated food and color additives. However, this document contained only minimum reference to neurotoxicity as a specific toxicological concern and only general mention of the types of data that should be collected to detect and assess adverse changes to the nervous system. The general nature of the toxicological information typically derived from studies based on the original Redbook has had only limited use as a guide for comprehensive assessment of neurotoxic hazard. This limitation is one of the issues being addressed in the current efforts to update the information provided in the Redbook. In the revised Redbook, neurotoxicity, encompassing adverse structural and functional changes to the nervous system, is explicitly identified as an important criterion in the assessment of food chemical safety. The proposed strategy for evaluating neurotoxic hazard has a tiered testing approach. Accordingly, testing would initially involve the identification of chemicals presumptively associated with neurotoxic effects. As appropriate, subsequent testing would be carried out to confirm and delineate the scope of the neurotoxicity, to determine the dose response kinetics, and to define the no-adverse-effect levels.

Prolonged alterations in canine striatal dopamine metabolism following subtoxic doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 4'-amino-MPTP are linked to the persistence of pyridinium metabolites.

Single toxic doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).HCl (2.5 mg/kg i.v.) and 4'-amino-MPTP.2HCl (22.5 mg/kg) induce loss of striatal dopamine (DA) and tyrosine hydroxylase (TH) activity and of nigral DA neurons in the dog. To examine the subacute neurochemical changes induced by low doses of MPTP and 4'-amino-MPTP, dose-response studies of these compounds were carried out in the dog, using 6- and 3-week survival times for these two compounds, respectively. Low single doses of MPTP (1.0, 0.5, and 0.1 mg/kg i.v.) and 4'-amino-MPTP (15, 7.5, and 3.75 mg/kg i.v.) did not cause depletion of canine striatal DA or TH or a loss of nigral neurons. However, levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased in a dose-related fashion, with significant loss of DOPAC being evident 6 weeks after the lowest administered dose of MPTP and 3 weeks after 4'-amino-MPTP. This selective loss of DA metabolites following nontoxic doses of MPTP and 4'-amino-MPTP led to a shift in the ratio of DA to DOPAC or HVA, which was characteristic for each compound. The measurement of striatal 1-methyl-4-phenylpyridinium (MPP+) and 4'-amino-MPP+ levels revealed that high concentrations (up to 150 microM) persist in the striatum for weeks following administration of a single nontoxic dose of MPTP or 4'-amino-MPTP. A causal relationship between the striatal concentration of MPP+ or 4'-amino-MPP+ and the change in DA metabolism as reflected in the DA/DOPAC ratio is suggested by a significant correlation between these measures. It is suggested that presynaptic sequestration and retention of MPP+ and 4'-amino-MPP+ by striatal DA terminals result in the inhibition of the monoamine oxidase contained within these terminals.

Extracellular concentrations of amino acid transmitters in ventral hippocampus during and after the development of kindling.

This study examined the possible involvement of amino acid release from ventral hippocampus in the establishment and maintenance of kindling in rats. Release of amino acids from ventral hippocampus was measured by microdialysis coupled with high-performance liquid chromatography. Samples were obtained by microdialysis perfusion of freely moving animals receiving deep prepiriform cortex (DPC) electrical stimulation. Samples of perfusate were collected before, during and after kindling was established. DPC kindling stimulation significantly increased concentrations of glutamate (Glu) and glycine (Gly) in perfusate from ventral hippocampus during kindling. Increased basal release of Glu was evident up to 30 days after the last electrical stimulation. We conclude that release of Glu and Gly in the ventral hippocampus may play an important role during establishment, but not in maintenance of kindling.

High doses of aspartame have no effects on sensorimotor function or learning and memory in rats.

Acute or repeated (14 days) intragastric administration of L-d-aspartyl-L-phenylalanine methyl ester (aspartame) suspended in saline and Tween-80 in doses of up to 1,000 mg/kg had no significant effect in male Fischer-344 rats on routine measures of sensorimotor function, including spontaneous motor activity, acoustic startle reflex and prepulse inhibition. Other experiments found that aspartame (500 or 1,000 mg/kg) had no significant effect on acquisition of passive or active avoidance or a spatial, reference memory task in the Morris water maze. A series of separate studies found that aspartame had no effects in rats fasted 24 hours prior to testing, or if it was suspended in carboxymethylcellulose or administered by the intraperitoneal route. Under the conditions of these experiments, large doses of aspartame have no significant neurobiological effects in adult rats as measured by procedures known to be sensitive to the neurobiological effects of neurotoxicants, including convulsants, organochlorine insecticides and heavy metals.

Alterations in acetylcholine-induced stimulation of inositol phospholipid hydrolysis in the dorsal hippocampus of kindled rats.

Agonist-induced turnover or release of inositolphosphates (IP) was studied in the dorsal and the ventral hippocampus 24 h, 1 month, and 3 months after the last electrical stimulus of kindled rats. A significant increase in acetylcholine (ACh)-stimulated IP turnover was observed in dorsal, but not ventral, hippocampus 24 h and 1 month after the last electrical stimulus. However, this effect was not evident 3 months after kindling. The excitatory amino acids (quisqualic acid and ibotenic acid) at the concentrations used, however, failed to produce any change in receptor-stimulated release or turnover of IP. Thus the changes in ACh-induced IP release, although long-term, are not permanent and do not appear to be released to the neurobiological alterations associated with the long-term maintenance of the kindling phenomenon.

Systemic administration of kainic acid increases GABA levels in perfusate from the hippocampus of rats in vivo.

The ventral hippocampi of male, Fischer-344 rats were implanted with microdialysis probes and the effects of systemically administered kainic acid (KA) (8 mg/kg, s.c.) on the in vivo release of amino acids were measured for four hours after administration. In order to measure GABA release in vivo, gamma-vinyl-GABA (GVG), an irreversible inhibitor of GABA transaminase, was injected intrahippocampally prior to perfusion. GVG pretreatment resulted in measurable levels of GABA in the perfusate without significant effects on the release of aspartate, glutamate, glutamine, glycine or taurine. Following GVG pretreatment systemic administration of KA produced a time-dependent increase in GABA, as well as all other amino acids except glutamine, which was initially decreased. These results show for the first time that systemically administered KA increases extracellular GABA levels, an effect previously reported only in vitro. These data suggest that prior to destruction of GABA-containing interneurons in the hippocampus, there is an increased activity of those GABA interneurons reflected as an increase in extracellular GABA levels.

Immunomodulatory effects of footshock in the rat.

Intermittent inescapable footshock (IIFS) treatment, administered to 3-month-old male rats, resulted in analgesia as well as discrete immunological and endocrine changes. The splenic lymphocyte proliferative response to concanavalin A (ConA) and phytohemagglutinin (PHA) was decreased by 20% and 41%, respectively. The primary IgM plaque forming cell (PFC) response to sheep red blood cells (SRBC), however, was not altered by IIFS administered either immediately or 24 h after injection of SRBC. IIFS also produced a significant (20-fold) increase in plasma corticosterone (CORT) as compared to non-shocked controls. The shock-induced suppression of splenic lymphocyte mitogenic response to PHA was blocked by 10 mg/kg naltrexone (NTX) administered immediately before IIFS. NTX alone had no effect on this mitogen response. However, NTX significantly attenuated the shock-induced rise in CORT even though NTX alone significantly elevated CORT in the non-shocked controls. These data suggest that IIFS alters cellular immune response but not the primary IgM PFC response (a measure of humoral immune function) and that the immunomodulatory effects may involve both an opioid and a corticosteroid component with respect to alterations in the splenic lymphocyte mitogenic response to PHA.

Food and Drug Administration: approach to evaluating neurotoxicity.

One of the primary features of the mission of the Food and Drug Administration is to assure with reasonable certainty that no harm will result from the intended use of chemicals added to food. In carrying out this mission the FDA uses a structured process to assess the safety of each food chemical. The detailed mechanics of this process are described in an FDA document entitled "Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Use in Food" (Food and Drug Administration, 1982). Central to its evaluation the FDA requires a collective set of information, including estimates of the anticipated human exposure to the food chemical and a broad-based toxicological profile. Certainly, any adverse effect observed in the nervous system is recognized as an important element in the determination of chemical safety and follow-up information which would enable an assessment of such an effect to be included in the toxicological profile. The agency's current approach to evaluating neurotoxicity should be viewed within the context of its overall strategy for the safety assessment of food chemicals. Four basic premises underlie the FDA's approach to safety assessment.

Oral administration of aspartame is not proconvulsant in rats.

These experiments examined the potential for single or repeated doses of aspartame to exacerbate or facilitate the production of seizures in Fischer-344 rats. In adult animals, 1,000 mg/kg of aspartame given by gavage acutely or over a 14 day period had no significant effect on the rate of kindling induced by stimulation of the prepyriform cortex. A single dose of 1,000 mg/kg of aspartame had no effect on the number of animals developing tonic seizures after electroconvulsive shock, nor did aspartame affect the frequency or duration of seizure activity after pentylenetetrazol. In a second series of studies, young male and female rats were dosed with 1,000 mg/kg of aspartame on day 3-13 or 21-35 of age. Prior exposure to aspartame had no significant effect on the rate of kindling at 90 days of age. These experiments indicate that aspartame does not act a pro-convulsant in rats.

Neurobehavioral effects of the calcium ionophore A23187.

The divalent cationic ionophore A23187 (calimycin) facilitates the transport of calcium ions across biological membranes, resulting in an increase of cytosolic calcium. A23187 has been used extensively in vitro to activate calcium-dependent neurocellular processes. Because of its potential usefulness as a neurotoxicological probe, our laboratory conducted a series of studies to characterize the neurofunctional consequences of A23187 in the intact organism. In addition to approximating the LD50, the effects of acute parenteral administration of A23187 on conditioned avoidance, nociceptive shock threshold, open-field activity, consummatory behavior, body temperature and neuromotor function, including general activity, coordination, balance and grip strength, were assessed in the rodent. The LD50 of A23187, administered intraperitoneally to adult male rats, was 9.2 mg/kg. The predominant overt signs of toxicity included lethargy, limb weakness and apnea. Lower doses, from 0.5 to 0.03 mg/kg, produced a variety of more subtle neurobehavioral effects, including a selective depression of motor activity, a moderate elevation of shock threshold, altered conditioned avoidance behavior and hypothermia.