Carboxyhemoglobin formation due to carbon monoxide exposure in rats.

The Coburn-Forster-Kane equation (CFKE) is a well-tested model for prediction of carboxyhemoglobin (COHb) formation due to carbon monoxide (CO) exposure in humans. There have been few and relatively poorly tested attempts to implement a CFKE for rats. Such an implementation is of interest because many experiments on the effects of CO in rats were done without measuring COHb. To extrapolate from rats to humans requires a rat version of the CFKE. Rats were exposed to 150, 250, 500, and 1000 ppm CO for up to 240 min. Blood gases and COHb were measured. A CFKE was implemented for rats by using parameters found in the literature and estimating them from the data. It was deduced from the blood-gas data that rats hyperventilate slightly as COHb increases. The blood-gas data were used to estimate ventilation and alveolar capillary oxygen partial pressure. The hyperventilation required an iterative solution to the CFKE. The iterative CFKE predictions were found to differ statistically from observations, but in explainable ways and/or in small amounts.

Behavioral toxicology and risk assessment.

In order to establish safe exposure levels to toxic chemicals, risk assessment guidelines have been developed. These guidelines evaluate epidemiologic and animal research data on a particular chemical, as well as dose-response relationships, animal to human extrapolation and assessment of exposure levels of populations. Using the guidelines, risk characterization is established in order to determine a strategy for reducing undesirable risk to human populations. Using both human neonatal lead exposure data and results from rodent and primate studies, this review examines the possibility that behavioral measurements are sufficient to provide adequate risk assessment guidelines for lead intoxication of the developing organism. The overall trend in these data during the past 10 years has been to show that exposures to inorganic lead at levels previously considered safe have long-lasting significant alterations in behavioral measures, suggesting that central nervous system function has been altered irreversibly. The conclusion is drawn that behavioral toxicology can provide sensitive, quantitative and reliable data for risk assessment and that in the future these methodologies could be used to set exposure guidelines for other neurotoxic chemicals.

Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats.

On day 15 of gestation, pregnant Sprague-Dawley rats were orally treated by gavage with 8 mg/kg of methyl mercury (MMC). At day 1 of postnatal life the levels of MMC in whole brain of exposed pups were found to be about 100 times higher than those of saline-exposed rats, while they were near to the control values at 21 days and practically normal at 60 days of age. Behavioral experiments showed that exposure to MMC in late gestation did not affect at any tested time (14, 21 and 60 days) locomotor activity or development of ultrasonic vocalization. An increased response to a challenge dose of amphetamine was, however, detected in MMC-exposed pups at day 14. This phenomenon was no longer evident at day 21 and 60 of age. In parallel, an increased density of dopamine receptors was found in the striatum at 14, but not at 21 and 60, days of age. From these data, we tentatively suggest that a high level of MMC induces a transient phenomenon of disuse-supersensitivity of the dopaminergic system. Moreover, further evidence that acute MMC exposure during prenatal life might induce permanent disturbances in learning and memory which could be partially related to a reduced functional activity of the glutamatergic system is provided.

Repeated exposure to diisopropylfluorophosphate (DFP) produces increased sensitivity to cholinergic antagonists in discrimination retention and reversal.

This experiment examined the effects of repeated exposure to diisopropylfluorophosphate (DFP), an organophosphate anticholinesterase, on the retention and reversal of a visual discrimination and on the number of muscarinic receptors in the brain. Rats were trained in a serial reversal procedure. After achieving stable performance, the rats were divided into two groups. One group received repeated injections of DFP, the other group received injections. To determine whether DFP-treated rats would be more sensitive than normal rats to stresses on the cholinergic system, each rat was injected with saline or one of three doses of scopolamine, a muscarinic receptor blocker, prior to testing on every 6th day. DFP alone caused no impairment in performance. Scopolamine produced a greater impairment in DFP-treated rats than in control rats. Similar results were obtained in a second behavioral task, match-to-sample in a water maze, using the same DFP treatment protocol and only one dose of scopolamine. The number of muscarinic receptors and acetylcholinesterase activity levels were reduced on the 2nd and 15th day after the end of DFP treatment. These results demonstrate that although repeated exposure to organophosphate anticholinesterases may not alter discrimination behavior directly, it may compromise the central nervous system so that it cannot react normally when challenged.

Mechanisms of neurotoxicity and their relationship to behavioral changes.

In this review some of the evidence relating behavioral alterations induced by 2 neurotoxic chemicals, lead acetate and methyl mercury is presented with an attempt to relate these changes to the underlying neurobiological mechanisms. In the case of neonatal lead poisoning, the results of the early behavioral studies were confounded by excessive lead concentrations resulting in undernutrition of the pups. Subsequent studies in both rodents and monkeys have shown that blood-lead concentrations comparable to those seen in children can induce behavioral alterations that may be related to hippocampal damage. In the case of methyl mercury which is a potent cytotoxic agent, prenatal exposure results in widespread cortical, and cerebellar alterations characterized by reduced myelination, delayed migration and loss of neurons. These morphological alterations are accompanied by permanent alterations in learning and memory as well as altered pharmacological sensitivity in catecholaminergic systems. Recommendations are made for better formulated behavioral and neurobiological assays in neurotoxicology in order to lead to a better understanding of the toxicity of chemicals.

The use of pharmacological challenges in behavioral toxicology.

Pharmacological challenges offer an opportunity to extend the behavioral observations into biological mechanisms. Behavioral measures may be used to generate hypotheses regarding the mechanisms of toxicity of chemicals, and pharmacological challenges may then be used to test and extend these hypotheses. In using d-amphetamine as a pharmacological probe it was possible to demonstrate postnatal effects of prenatal exposure to methyl mercury (MMC), which were absent without the challenge. This suggests catecholaminergic function to be altered by MMC. Additionally, the use of stressful situations often allows the unmasking of latent damage to the nervous system, which otherwise may remain covered by the functional reserve capacity of the brain.

Complex maze performance during carbon monoxide exposure in rats.

Most human victims of residential fires die of smoke inhalation. The cause of death of the victims is attributed to high levels of carboxyhemoglobin, but it is not clear why the victims are unable to escape even from locations remote from flaming combustion. In an attempt to provide a model of escape from toxic gases using animals, a complex maze was built for rats with 8 choice points. The animals were 24 hr water deprived and trained to remain in the start box for 15 min. Following this period, a rat was released in the maze and had to learn to avoid blind alleys and reach the goal box for water reinforcement within 15 min. Total time to traverse and total distance in the maze were recorded. Each animal was given one trial per day. After stable running times were established, different groups of six rats were exposed to 2000, 3000, 3500, and 4000 ppm of carbon monoxide (CO) when placed in the maze. Each animal was exposed to CO only once. On the day after CO exposure the rats were implanted with an arterial cannula and on the next day each animal was exposed to the same CO concentration it had previously experienced for 30 min. Blood samples were taken every 5 min. The effect of increasing CO concentrations was to increase maze running times as well as to decrease the number of animals reaching the goal. At 3500 ppm no animal reached the goal. At 2000 ppm, the animals that failed to reach the goal moved a greater distance than animals that reached the goal.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical interactions of carbamates and ecothiophate with the activated conformation of nicotinic acetylcholine receptor.

Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with Ki values of 10, 20, and 1,600 microM, respectively. The inhibition was mixed competitive-noncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6, 50, and 1,000 microM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-alpha-bungarotoxin ([125-I]-alpha-BGT) with the following decreasing order of potency: Neo less than Physo less than Eco less than Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-alpha-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-alpha-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites.

Behavioral consequences of intraperitoneal carbon monoxide administration in rats.

Blood carboxyhemoglobin (HbCO) was determined 15, 30, 45, 60, 90, and 120 min following ip injection of 2.5, 5, 10, 20, and 40 ml pure carbon monoxide (CO)/kg body wt in rats. These CO doses produced HbCO concentrations of 12, 24, 35, 45, and 60%, respectively, at 30 min postinjection. Once these normative data were obtained, a group of eight naive rats were trained to produce a rapid sequence of responses in a stimulus-tracking task, then they were exposed to each of the doses of CO. The 10-ml/kg dose produced a slight decrement in performance, the 20-ml/kg dose reduced correct responses by nearly half, and the 40-ml/kg dose resulted in virtually complete cessation of responding. CO exposure resulted in longer pauses in responding with increasing dose, but the distribution of errors produced in the stimulus-tracking task remained relatively parallel across the range of CO exposures. Thus CO exposure impaired tracking performance but had relatively little effect on the pattern of errors rats produced during the stimulus-tracking test. Taken together these results contradict previous reports purporting to show that CO by ip administration has no behavioral effects; instead, the results indicate that CO administration via the ip route has very similar effects to inhaled CO on behavior.

Hypothalamic monoamine metabolism in mice: evaluation of drug challenges and neurotoxic insult.

Hypothalamic monoamine metabolism was evaluated in male and female mice that were administered monosodium glutamate (MSG, 4 mg/g) on postnatal day 4. Hypothalamic dopamine (DA) content was reduced approximately by 22% across all experiments and pituitary DA content was also significantly decreased. Despite reductions in hypothalamic DA levels, MSG-treated mice did not exhibit significant reductions in the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Hypothalamic monoamine and metabolite profiles in MSG-treated and control mice were examined after the administration of pargyline (PAR), alpha-methyl-p-tyrosine (MPT) or reserpine (RES). DA turnover as indexed by PAR and MPT techniques were not as conclusive as the metabolite measures in suggesting an accelerated DA utilization. MSG-treated mice were more resistant to the DA-depleting actions of reserpine than were the controls. No alterations were found in hypothalamic norepinephrine metabolism in MSG-treated mice, however, after drug challenges, alterations were found in serotonin metabolism. These results indicate the extent and nature of the changes that occur in hypothalamic monoamine metabolism after neonatal MSG treatment. The DA neurons that survive MSG treatment appear to exhibit neurochemical characteristics dissimilar to those of tuberoinfundibular DA neurons. Additionally, the neuropharmacological actions of MPT, PAR and RES are discussed as they relate to hypothalamine monoamine metabolism in the mouse.

Neonatal monosodium glutamate administration alters noradrenergic measures in the brainstem of the mouse.

Mice treated neonatally with MSG (4 mg/g) were compared to saline-injected controls on a number of neurochemical parameters of brainstem noradrenergic activity. MSG treatment resulted in an attenuation of brainstem norepinephrine (NE) decline after alpha-methyl-p-tyrosine administration. Neonatal MSG administration did not result in alterations in the steady state levels of brainstem NE or MOPEG. The synthesis of NE was slightly increased in the pons-medulla of MSG-treated mice as indexed by pargyline-induced NE accumulation. NE release, however, appeared diminished as reflected by a significant (p less than 0.05) decrease in the ratio of normetanephrine to NE found in the pons-medulla of MSG-treated mice given pargyline. The results suggest that MSG-induced damage to the arcuate nucleus produces selective alterations in brainstem NE systems. These alterations may reflect the toxic action of MSG on the opiomelanocortin neurons of the arcuate nucleus or other descending systems that are damaged by MSG. The loss of the descending opiomelanocortin input to the brainstem could result in these types of neurochemical consequences since the pharmacologic action of opiate drugs results in a selective enhancement of brainstem NE turnover in rodents.

Tuberohypophyseal and tuberoinfundibular dopamine systems exhibit differential sensitivity to neonatal monosodium glutamate treatment.

The arcuate nucleus (AN) is the presumed origin of the dopaminergic innervation of posterior lobe of the pituitary. Posterior lobe dopamine levels were determined in rats that had been neonatally treated with monosodium glutamate (MSG) to lesion the AN. MSG-induced AN damage was confirmed neurochemically, histologically and immunocytochemically. MSG treatment resulted in a substantial loss of AN neurons and approximately a 50% loss of dopamine uptake capacity (Vmax) in the mediobasal hypothalamus. Posterior pituitary dopamine levels were not significantly altered by MSG-induced AN damage. These results suggest that MSG treatment spares the tuberohypophyseal dopamine system and that the AN may not be the sole origin of the dopaminergic innervation of the posterior pituitary.

Behavioural and neurochemical changes in offspring of rats exposed to methyl mercury during gestation.

On day 8 of gestation, pregnant Sprague-Dawley rats were intubated with 8 mg/kg of methyl mercury (MM). At 15 days of age, stereotyped sniffing was elicited by a challenge dose of apomorphine (1 mg/kg) only in pups prenatally exposed to MM; furthermore, at 22 days of age, the stereotyped behaviour induced by apomorphine (0.5-1 mg/kg) was significantly potentiated in MM-pretreated animals. Neurochemical data showed that at 22 days of age there was a significant increase of 3H-spiroperidol binding sites in striatal membranes of MM-pretreated rats. At 40 and 60 days of age, these behavioural and neurochemical changes were absent. On the other hand, prenatal exposure to MM influenced neither the effects of a challenge dose of clonidine (0.025 mg/kg) on locomotor activity nor the affinity and the density of alpha-2 adreno-receptors in cortex of 15, 22, 40 and 60 day old rats. The behavioural alterations in the response to apomorphine presumably result from an enhancement of dopamine binding sites induced by MM. Finally, our data further confirm that prenatal MM induces long lasting behavioural alterations which can be accurately displayed by using passive avoidance procedures.

Chronic lead administration in neonatal rats: electron microscopy of the retina.

The morphologic effects on the retina resulting from chronic lead exposure were assessed in neonatal rats. Newborn rats nursed from dams were given a low (0.115%) or a high (4.5%) concentration of lead in their diet. At day 21 the pups were weaned to the mother's diet. The retinas of the pups were studied by electron microscopy at various ages up to day 60. High and low lead concentrations produced necrosis of photoreceptor cells and cells of the inner nuclear layer. The high lead concentration, in addition, was associated with swelling of endothelial cells of the retinal vessels and narrowing of the lumen. Increased permeability of the retinal vessels and pigment epithelium to horseradish peroxidase was also observed under the high-dose condition. The authors conclude that lead can produce direct neuronal damage and, at high doses, produces retinal vascular lesions and alteration of the blood-retinal barrier.

Effects of chronic lead exposure on release of endogenous catecholamines: a preliminary communication.

The release of endogenous dopamine and norepinephrine from six brain regions (cortex, hippocampus, hypothalamus, striatum, s. nigra, n. accumbens-o. tubercle) of chronically lead-exposed rats was compared to release from controls. Prenatal and postnatal chronic lead intoxication was induced by 1000 ppm lead acetate in the drinking water of dams 70 days prior to mating and throughout gestation and lactation. Offspring were also maintained on 1000 ppm lead acetate until sacrificed at approximately 60 days of age. Release was measured in vitro from tissue minces prepared from both lead-exposed and control rats. There were no significant differences in spontaneous or potassium-stimulated norepinephrine release between lead-exposed and control rats. Potassium and amphetamine both stimulated striatal dopamine release; however, no marked differences between lead-exposed and control tissue were seen. Potassium-stimulated release of dopamine from the nucleus accumbens-olfactory tubercle area was increased in the lead-exposed animals. This is interesting in light of the postulated role of the n. accumbens in the central regulation of motor activity and the reported alterations in activity in lead-exposed rodents.

Regional brain and pituitary cyclic nucleotide response following central cholinergic stimulation in rats chronically exposed to lead.

Long Evans rats were exposed continuously to lead (1,000 ppm of lead acetate in the drinking water of dam or weanling) throughout gestation, lactation and until sacrifice at 60 days of age. A matched group of control animals was exposed to distilled water. On the day of sacrifice, control and lead treated animals were administered methylatropine 40 min. and oxotremorine (a muscarinic agonist) 10 min. prior to sacrifice by microwave irradiation. Locomotor activity was monitored during the 10 min. immediately preceding sacrifice. Levels of cyclic AMP and cyclic GMP were determined in 21 brain regions by radioimmunoassay. Locomotor activity following oxotremorine was significantly reduced in both lead treated and control animals. Levels of cyclic GMP were increased in septal region and frontal cortex and reduced in cerebellum and inferior colliculus of both groups of animals. The level of cyclic AMP in the pituitary increased 20-25 fold with a greater response noted in lead treated rats. Elevations of cyclic AMP were also noted in the interpeduncular nucleus, corpus striatum and substantia nigra in both groups. These data suggest that the cellular response to a muscarinic agonist was significantly affected by chronic exposure to lead in the pituitary but not in the remainder of the central nervous system.

Adenosine receptor interactions and anxiolytics.

[3H]-N6-cyclohexyladenosine and [3H]-1,3-diethyl-8-phenylxanthine label the A1 subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A1 adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N6-(phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and N6-(L-phenylisopropyl)adenosine cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately. Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine.

Interactions in the behavioral effects of methylxanthines and adenosine derivatives.

The role of adenosine receptors in behavioral effects of alkylxanthines was evaluated in mice. The relative potencies of alkylxanthines in reversing locomotor activity depression elicited by L-phenylisopropyladenosine (L-PIA) were similar to relative potencies in competing for adenosine receptors labeled by [3H]cyclohexyladenosine. Whereas L-PIA at i.p. doses of 0.10 mumol/kg and higher depressed locomotor activity, lower doses (0.01 and 0.05 mumol/kg) augmented locomotor activity. At the doses evaluated, caffeine did not further augment the L-PIA (0.05 mumol/kg)-enhanced locomotor activity. Low doses of diazepam, like L-PIA, augmented locomotor activity. Combining locomotor depressant doses of diazepam and caffeine produced a paradoxical stimulation of activity, as observed also for L-PIA and caffeine. Low doses of diazepam but not L-PIA increased crossings between the light and dark sites in a shuttle box, indicating a difference in the behavioral profile of these two agents. At behaviorally effective doses, L-PIA did not alter blood pressure or heart rate, but elicited some premature ventricular contractions which, however, occurred to a similar extent at locomotor depressant and stimulant doses of L-PIA. Brain levels of L-PIA at the lowest behaviorally active doses were adequate to occupy more than 50% of adenosine receptors. Thus, the behavioral effects of L-PIA appeared to be mediated in the brain and were not secondary to the cardiovascular effects.