Effects of subacute toluene inhalation on its metabolism and disposition in rat.

Exposure of adult male rats to 300 ppm of toluene for 1 to 15 weeks 6 h daily caused accumulation of the solvent in brain and in perirenal fat. The body solvent content tended to decrease during extended exposure which might be explained by the enhanced activity of drug-metabolizing enzymes in liver. Our findings also indicate that metabolic and functional adaptation takes place in longer exposures to low levels of toluene vapour.     

Effects of toluene inhalation on brain biogenic amines in the rat

The effects of toluene exposure on the biogenic amine concentrations in the central nervous system were investigated in the rat. Toluene was administered via inhalation to groups of rats at concentrations of 0, l00, 300, or 1000 ppm. After an 8-h continuous exposure, animals were sacrificed and whole brain concentrations of dopamine (DA), norepinephrine (NE), and 5-hydroxytryptamine (5-HT) were determined. The data indicated a significant increase in whole brain concentrations of DA following the 100-ppm exposure. A regional analysis of DA, NE, and 5-HT concentrations in rats exposed to 1000 ppm of toluene for 8-h indicated a significant increase in DA concentration in the striatum. A significant increase in NE concentrations was detected in the medulla and midbrain while 5-HT concentrations were significantly increase in the cerebellum, medulla, and striatum. The results indicate that toluene action results in elevated concentrations of behaviorally significant neuro-transmitters.     

Acute toluene exposure and rat visual function in proportion to momentary brain concentration.

Acute exposure to toluene was assessed in two experiments to determine the relationship between brain toluene concentration and changes in neurophysiological function. The concentration of toluene in brain tissue at the time of assessment was estimated using a physiologically based pharmacokinetic model. Brain neurophysiological function was measured using pattern-elicited visual evoked potentials (VEP) recorded from electrodes located over visual cortex of adult male Long-Evans rats. In the first experiment, VEPs were recorded before and during exposure to control air or toluene at 1000 ppm for 4 h, 2000 ppm for 2 h, 3000 ppm for 1.3 h, or 4000 ppm for 1 h. In the second experiment, VEPs were recorded during and after exposure to clean air or 3000 or 4000 ppm toluene. In both experiments, the response amplitude of the major spectral component of the VEP (F2 at twice the stimulus rate in steady-state responses) was reduced by toluene. A logistic function was fit to baseline-adjusted F2 amplitudes from the first experiment that described a significant relationship between brain toluene concentration and VEP amplitude deficits. In the second experiment, 3000 ppm caused equivalent VEP deficits during or after exposure as a function of estimated brain concentration, but 4000 ppm showed a rapid partial adaptation to the acute effects of toluene after exposure. In general, however, the neurophysiological deficits caused by acute toluene exposure could be described by estimates of the momentary concentration of toluene in the brain at the time of VEP evaluation.     

An electrophysiological analysis of rat ventral tegmental dopamine neuronal activity during acute toluene exposure.

Inhalant abuse is a common, potentially lethal, form of drug abuse. Although the putative psychotropic component of some popularly abused inhalants appears often to be the organic solvent toluene, its effects on midbrain neurones which comprise reward pathways have not been established. Therefore, the present study was designed to assess the response of ventral tegmental dopamine neurones during toluene inhalation. Electrophysiological determinations were made using extracellular single-unit recordings in ketamine anaesthetized rats that were exposed to acute (1-15.3 min.) concentrations of toluene vapor (11,500 ppm) similar to those consumed by inhalant abusers. Toluene exposure through a tracheal breathing tube elicited two distinctly different patterns of response in dopamine neurons. One pattern consisted of an initial stimulation of neuronal firing (+221%+/-72%; <8.5 min.) followed by an attenuation of the firing rate with continued exposure (+58.7%+/-6.3%; >8.5 min.). The other pattern consisted of only an inhibition of firing regardless of the length of exposure. Furthermore, the changes in firing rates were paralleled by changes in number of action potentials contained in bursts. Blood samples taken at the time of the dopamine recordings revealed comparable toluene concentrations (4-79 microg/ml, n=24) regardless of the patterns of response. These results suggest that mesolimbic dopamine neurotransmission can be changed by an exposure paradigm comparable to that used by human abusers, and that these changes may be integral to the reinforcing effects underlying inhalant abuse.     

Effects of emopamil on postischemic blood flow and neuronal damage in rat brain

Summary The effects of the calcium entry blocker emopamil on physiological variables, local cerebral blood flow (LCBF) and on hippocampal cell damage were evaluated after 10 min of forebrain ischemia in the rat. LCBF was determined with the ¹⁴C-iodoantipyrine technique after 2, 10, and 60 min of postischemic recirculation. Histological evaluation was performed 7 days after ischemia in cortical and hippocampal tissue by determination of the percentage of necrotic neurons. Preischemic application of emopamil [4 mg/kg racemate or 2 mg/kg (S)-emopamil; i.v.] caused increases in LCBF in cortical areas but did not alter blood flow in the hippocampus at 2 min of recirculation. After 10 and 30 min of flow resumption no differences in LCBF between drug-treated and control animals were observed. In the histological series (S)-emopamil was applied at doses of 2, 4 or 6 mg/kg before the induction of ischemia. After 7 days of postischemic recovery, neuronal damage was significantly reduced by the calcium antagonist in hippocampal CA 1 sector at all doses tested, the most prominent effects being observed with the lowest dose. At this dose cell loss in the Ca3 sector was also reduced. In cortical tissue the number of necrotic cells remained unchanged by emopamil treatment. It is concluded that the calcium antagonist emopamil can reduce ischemia-induced neuronal cell damage. The compound improves circulation in cortical tissue only during early recovery but not at later phases of reflow, i.e. the period of delayed hypoperfusion. These increases in blood flow are not of crucial importance for ultimate neuronal death in this area. The ameliorative action of emopamil on the survival of hippocampal neurons is not associated with blood flow changes and therefore seems to reflect a direct effect on cerebral parenchyma. Send offprint requests to G. W. Bielenberg at the above address     

Persistent effects of neonatal toluene exposure on regional brain catecholamine levels and turnover in the adult male rat

Effects of neonatal toluene exposure (80 ppm, day 1-7, 6 h/day) have been studied on regional brain catecholamine levels and utilization, and on serum levels of hypophyseal and adrenocortical hormones in the adult male rat. Catecholamine levels were measured by quantitative histofluorimetry in the forebrain and hypothalamus and by high pressure liquid chromatography with electrochemical detection in the substantia nigra. Catecholamine utilization was evaluated from the decrease in catecholamines seen after tyrosine hydroxylase inhibition using alpha-methyl-p-tyrosine methyl ester hydrochloride (alpha MT, 250 mg/kg, i.p., 2 h). Serum levels of thyroid stimulating hormone, corticosterone, aldosterone, prolactin and luteinizing hormone were measured by radioimmunoassays. Neonatal toluene exposure produced a reduction of dopamine levels and utilization selectively in the olfactory tubercle and substantia nigra of the adult rat. Furthermore, neonatal toluene exposure produced a significant reduction in the noradrenaline levels and utilization in the substantia nigra and an increase of noradrenaline utilization selectively in the subependymal layer of the median eminence and of the magnocellular part of the paraventricular hypothalamic nucleus. The serum hormone levels were not significantly influenced by neonatal toluene exposure as evaluated in adulthood. However, the alpha MT induced increase in serum prolactin levels was reduced following neonatal exposure to toluene. Neonatal toluene treatment was also found to alter the responses of the catecholamine neurons to subacute toluene exposure in adulthood. In some of the dopamine nerve terminal systems of the forebrain and in the dopamine cell body containing area of the substantia nigra neonatal toluene exposure appears to have made the dopamine neurons insensitive to adult subacute toluene exposure. In the hypothalamic noradrenaline nerve terminal systems, there were even reversed responses to subacute toluene exposure. The present results indicate that neonatal toluene exposure in doses at the threshold limit value produces persistent changes in dopamine and noradrenaline neurons of the forebrain, hypothalamus and substantia nigra in the presence of a relatively intact neuroendocrine system. In addition, neonatal toluene exposure appears to diminish or even counteract the responses to subacute toluene treatment in adulthood.     

Effects of postnatal ethanol exposure at different developmental phases on neurotrophic factors and phosphorylated proteins on signal transductions in rat brain

Exposure to ethanol during development induces severe brain damage resulting in a number of CNS dysfunctions including microencephaly and mental retardation in humans and in laboratory animals. The most vulnerable period to ethanol neurotoxicity coincides with the peak of brain growth spurt. Recently, neurotrophic factors and/or their signal transduction pathways have been reported as a potential relevant target for the developmental neurotoxicity of ethanol. The present studies were designed to investigate the effects of ethanol given in various developmental phases during the brain growth spurt in rats. Rat pups were assigned to the three treatment groups and treated with 5 g/kg of ethanol for three days, on postnatal days (PND) 2-4, 6-8 or 13-15. Whole brain weights were reduced only in the PND 6-8 group concurrently with the reduction of GDNF mRNA in cortex in this group. BDNF mRNA expression was reduced in both the PND 6-8 and 13-15 groups, while mRNA expressions of NT-3 and NGF were unchanged in all three groups. Phospho-Akt level was mostly reduced in the PND 6-8 group. Both phospho-MAPK and p-70S6 kinase levels were decreased in all groups whereas no changes were observed in either phospho-PKCzeta or CREB level. The phosphorylation of Akt was immediately inhibited after single administration of ethanol, and its inhibition was correlated with variations in blood ethanol concentration. These findings suggest that GDNF and the phosphorylation of Akt play a possible key role in the ethanol-induced developmental neurotoxicity.     

Perinatal lead exposure alters the expression of neuronal nitric oxide synthase in rat brain

Environmental exposure to lead (Pb) is known to affect the developing nervous system causing cognitive deficits in children. The diffusible nitric oxide (NO) is a biological messenger known to be involved in brain development. We examined the developmental changes of neuronal nitric oxide synthase (nNOS) in cerebellum and hippocampus of developing rat brain by radiometric assay, Western blot analysis and immunohistochemistry. Pb-exposure (0.2% Pb acetate) was initiated on gestation day 6 through the drinking water of the dam and continued through birth and postnatal days (PNDs) 1 to 21. The pups were never exposed to Pb directly. Pb exposure was stopped on weaning of pups from mothers on PND 21. The changes in nNOS were measured in the offspring on PNDs 7, 14, 21, and 35. The nNOS activity was increased gradually from PNDs 7 to 35 in both cerebellum and hippocampus of control rats when the enzyme activity was determined in the presence of either 0.5 or 6 microM calcium (Ca2+) in the reaction mixture. However, Pb exposure decreased the nNOS activity significantly at PNDs 21 to 35 as compared to respective controls when the enzyme activity was determined in the presence of 6 microM Ca2+. The decrease of nNOS was even greater and evident at all PNDs tested when the enzyme activity was assayed in the presence of physiological concentration of Ca2+ (0.5 microM). These findings were further strengthened by the in vitro studies. The cerebellar nNOS activity was inhibited much more at low Ca2+ (0.5 microM) as compared to 6 microM Ca2+, with IC50 values of 35 and 50 nM Pb, respectively. The nNOS protein levels and immunoreactivity in the cerebellum and hippocampus of rats perinatally exposed to Pb were decreased as compared to controls at PNDs 21 and 35. These data suggest perinatal Pb exposure decreases the nNOS in the developing brain. The decrease of nNOS activity and protein may explain the Pb-mediated cognitive deficits because NO regulates long-term potentiation (LTP) and other neurophysiological events in the developing nervous system.     

Effects of chronic toluene exposure on central monoamine and peptide receptors and their interactions in the adult male rat

The effects of chronic toluene exposure (CTE) (80 ppm, 6 h/day, 5 days/week, 3 months) were studied on neuropeptide and 5-hydroxytryptamine receptors, on protein phosphorylation levels and on catecholamine levels in various brain regions in the 15-month-old male rat. Behavioral parameters and serum levels of hypophyseal hormones and corticosterone were also analyzed. CTE selectively reduced [3H]neurotensin [( 3H]NT) binding in the basal layers of the orbital cortex. Instead, CTE increased the binding of [3H]etorphine in the nucleus accumbens and of [125I]vasoactive intestinal polypeptide [( 125I]VIP) in the area postrema and hypoglossal nucleus. Acute treatment with the irreversible monoamine receptor antagonist N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) increased the binding of [3H]NT in the orbital cortex in toluene exposed rats as compared with the reduced [3H]NT binding obtained in air exposed rats treated with EEDQ. Furthermore, the EEDQ induced increase in [125I]VIP binding in the area postrema and the hypoglossal nucleus was replaced by a reduced binding of [125I]VIP in EEDQ-treated CTE rats. CTE produced an overall increase in calcium-induced back phosphorylation and an overall decrease in cyclic adenosine monophosphate-induced back phosphorylation in the frontoparietal cortex. Noradrenaline stores tended to be reduced within various hypothalamic subnuclei and the serum prolactin levels were increased following CTE. However, no marked effects of CTE were seen on the behavioral parameters. In conclusion, the regional selectivity of CTE in disturbing [3H]NT and [125I]VIP binding may be due to the demonstrated vulnerability of monoamine-neuropeptide interactions to toluene.     

Ganglioside GM1 counteracts the enhancing effects of subacute toluene exposure on apomorphine-induced locomotor activity.

Previous studies indicate that subacute toluene exposure enhances the effects of postsynaptic doses of apomorphine on locomotor activity in the rat. We have now studied the effects of the ganglioside GM1 on toluene-affected apomorphine-induced (1 mg/kg, s.c.) locomotion, motility, and rearing. Treatment with GM1 (10 mg/kg, i.p., 1 h before exposure) was found to counteract or even reverse the enhancing effect of toluene on apomorphine-induced locomotion and rearing, but had similarly to toluene no significant effects on apomorphine-induced motility or on spontaneous locomotor activity. The antagonistic effects of GM1 may be due to its ability to block toluene-induced changes in D2 receptor binding.     

Behavioral effects of chronic toluene exposure in the developing rat

The effects of acute and chronic toluene exposure on the hypnotic effect, the righting reflex latencies and the blood and tissue toluene contents were studied in rats during development. The data showed a progressive significant prolongation of the hypnotic effect latencies until the third and fourth postnatal weeks, followed by a significant continuous declining trend until the eighth week postpartum. The measure of the righting reflex latencies followed an opposite temporal course compared to that of hypnotic effect measurements. The acute and chronic toluene exposure did not reveal significant differences in toluene concentrations of blood, brain and liver tissues. The data suggest that chronic toluene treatment may probably be inducing behavioral manifestations of a tolerance phenomenon combined with maturational influences in the developing rat.     

Effects of maternal toluene exposure on testosterone levels in fetal rats

The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17α-hydroxylase/c17-20 lyase, 17β-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3β-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 3β-HSD expression.     

Effects of toluene exposure on the spontaneous cortical activity in rats

Effects of toluene on the electroencephalogram (EEG) and its power spectra were measured during a 2-hr exposure in a dynamic inhalational chamber in rats. Rats were exposed to one of six graded concentrations (110.6, 162.5, 432, 676, 1558, 2730 ppm) of toluene on different days. It was found that the duration of waking (W) was increased with a decrease in duration of rapid eye movement (REM) sleep even at 110.6 ppm. Duration of nonrapid eye movement (NREM) sleep was decreased with an increase of W and a decrease of REM sleep at 162.5 ppm. Dose-related effects were noted in higher concentrations. The power of delta frequency band was increased with a decrease of theta frequency band power at hr 1 of exposure to 676 ppm during REM sleep recorded from the visual cortex. The power of theta frequency band was also decreased at hr 2 of exposure at 432 ppm. During W and NREM sleep power spectra were not changed significantly. Results indicate that the changes of EEG are a sensitive measure of the effects of toluene on the central nervous system (CNS).     

Effects of toluene exposure on the rest-activity cycle of rats

Rats were exposed to toluene at a concentration of 1000 ppm, 6 h/day, 6 times weekly, for 4 weeks. Functional disturbance of the circadian rhythm of spontaneous activity was found after repeated exposure to toluene, although single exposure under the same conditions did not influence the rhythm. The disturbance was characterized by a significant increase in distribution of activity in the ‘Light’ (L) period, and a significant relationship was observed between the total number of days of exposure and the activity level in the L period. The toluene concentration in the blood immediately after the exposure was 6.8–7.2 μg/ml after one and 18 days of exposure, respectively.     

Effects of the novel cyclosporine derivative PSC833 on glucose metabolism in rat primary cultures of neuronal and glial cells

Cyclosporine A (CsA) and the cyclosporine degrees congener PSC833 are known to cause transient CNS symptoms at high dosages in animal and man. Since impaired glucose metabolism plays a fundamental role in many heriditary and drug-induced neurological disorders, it was the purpose of the present study to evaluate whether this mechanism of pathogenesis might apply to PSC833 and CsA, using neural cells from rats. PSC833 and CsA were investigated in primary cultures of rat neuronal and glial cells at the concentration of 0.1, 1, 10, and 20 microM for 24 and 48 hr. Lactate dehydrogenase was determined as a marker of cytotoxicity. Cell proliferation was determined in astrocytes. Cellular glucose metabolism was investigated by 13C-NMR using [1-13C]glucose as a substrate. Glucose and lactate concentrations in the cell culture supernatants were determined spectrophotometrically. PSC833 at 10 microM was not cytotoxic in neuronal or glial cells nor did it inhibit proliferation in astrocytes 24 hr after incubation. Under the same conditions, the determination of [1-13C]glucose and [3-13C]lactate revealed significantly increased glucose consumption and lactate production in both cell types, as well as decreased levels of Krebs cycle intermediates. In the cell culture medium of both cell types after treatment with 10 microM PSC833, the rates of glucose consumption and lactate formation increased in comparison to controls, between 60-83% and 54-78%, respectively. PSC833 (10 microM) and CsA (20 microM) resulted in nearly similar increased glucose consumption and lactate production. The major PSC833 metabolite in rats, M9, which was devoid of CNS effects, did not cause significant changes in glucose metabolism. The present data suggest that PSC833-impaired tricarboxylic acid cycle activity, resulting in decreased Krebs cycle metabolites, can cause energy depletion and acidosis, which might contribute to the transient neurological symptoms of PSC833 and CsA.     

Effects of prenatal exposure to styrene on neurochemical levels in rat brain.

Styrene was evaluated to determine its neurochemical effects in the offspring of rats exposed during the gestation period. Maternal Wistar rats were exposed to 0, 50 or 300 ppm styrene during gestation days 6 to 20 and the neurochemical effects on their offspring were compared with their pair-feeding and ad lib. feeding controls. The cerebrum weights at birth on day 0 were significantly lower than those for an ad lib. feeding control group. Neurotransmitter analyses showed decreases of neuroamines, especially 5-hydroxytryptamine and homovanillic acid in the cerebrum of newborn offspring of dams receiving a 300 ppm styrene exposure compared with the ad lib. fed control group and homovanillic acid was also decreased compared to the pair-feeding control. On postnatal day 21, the styrene-exposure pups showed a significant decrease of 5-hydroxyindoleacetic acid in the frontal neocortex compared with the ad lib. control group. In the hippocampus a significant decrease of 5-hydroxyindoleacetic acid was observed compared with both control groups. Moreover, the ratio of 5-HIAA/5-HT in the hippocampus was significantly decreased among the styrene-exposure groups. The 50 ppm styrene exposed group induced increase of concentrations of 5-hydroxytryptamine in the striatum. These results suggest that prenatal styrene exposure affects the developing fetal brain in terms of a few signs of neurochemical alteration.     

Effects of short-term toluene exposure on ligand binding to muscarinic acetylcholine receptors in the rat frontal cortex and hippocampus

Changes in the binding affinity of the muscarinic acetylcholine receptor agonist carbamylcholine were determined in membranes isolated from the brains of rats exposed to toluene at concentrations of 500-2,000 ppm for 6 h. Membrane fractions of the frontal cortex and hippocampus were prepared and agonist-binding affinities were determined by measuring the displacement of [3H]N-methyl scopolamine-binding activity by carbamylcholine. In the frontal cortex, the affinity of high-affinity carbamylcholine binding was reduced following exposure to toluene at a concentration of 1000 ppm or higher. However, in the hippocampus, the affinity of high-affinity binding of carbamylcholine was increased following exposure to toluene. These observations suggest that toluene exposure affects binding affinity of carbamylcholine, and the effect differs by brain region.     

Effects of subchronic exposure to toluene on working and reference memory in rats

Rats that had inhaled 600 ppm of toluene vapor 24 h a day for 50 days after weaning at 3 weeks of age were trained in a radial-arm maze with a 4-out-of-8 baiting procedure, and their performance based upon reference and/or working memory was compared with that of air-exposed control animals during the early stage of acquisition. Pharmacological challenge testing was also conducted after completing a total of 48 training sessions; the effects of scopolamine and methylscopolamine on the maze performance were measured after acute IP administration to determine the long-lasting effects of toluene exposure. During the acquisition stage, toluene-exposed rats made a significantly smaller number of reference memory errors (entries into “never-baited” arms) and total arm entries than the control rats. No significant effects of exposure were observed for working memory errors (reentries into “already-entered” arms). During the pharmacological challenge testing, only scopolamine increased both types of errors significantly. No significant differences due to toluene exposure were revealed.