Prenatal and perinatal acrylamide disrupts the development of cerebellum in rat: Biochemical and morphological studies.

Acrylamide is known to cause neurotoxicity in the experimental animals and humans. The literature on its neurotoxic effect in the adult animals is huge, but the effect of acrylamide on the embryonic and postnatal development is relatively less understood. The present study examined its effects on the development of external features and cerebellum in albino rats. Acrylamide was orally administered to non-anesthetized pregnant females by gastric intubation 10 mg/kg/day. The animals were divided into three groups as follows. (1) Group A, newborn from control animals; (2) Group B; newborns from mothers treated with acrylamide from day 7 (D7) of gestation till birth (prenatal intoxicated group); (3) Group C; newborns from mothers treated with acrylamide from D7 of gestation till D28 after birth (perinatally intoxicated group). Acrylamide administered either prenatally or perinatally has been shown to induce significant retardation in the newborns' body weights development, increase of thiobarbituric acid-reactive substances (TBARS) and oxidative stress (significant reductions in glutathione reduced [GSH], total thiols, superoxide dismutase [SOD] and peroxidase activities) in the developing cerebellum. Acrylamide treatment delayed the proliferation in the granular layer and delayed both cell migration and differentiation. Purkinje cell loss was also seen in acrylamide-treated animals. Ultrastructural studies of Purkinje cells in the perinatal group showed microvacuolations and cell loss. The results of this study show that prenatal and perinatal acrylamide or its metabolites disrupts the biochemical machinery, cause oxidative stress and induce structural changes in the developing rat cerebellum.

A new murine model of giant proximal axonopathy.

The aromatic gamma-diketone 1,2-diacetylbenzene (1,2-DAB), the putative active metabolite of the organic solvent 1,2-diethylbenzene, forms blue-colored polymeric protein adducts and induces the formation of amyotrophic lateral sclerosis (ALS)-like giant, intraspinal neurofilamentous axonal swellings in Sprague Dawley rats. The pathogenetic mechanism of this neuropathy has yet to be understood. We assessed whether these pathological changes are also seen in the C57BL/6 mouse, the animal of choice for toxicogenomic studies. Mice were treated intraperitoneally with 30, 35, 50, or 70 mg/kg 1,2-DAB or its inactive isomer 1,3-DAB per day (or on alternate days) for up to 43 days. Animals treated with 30 or 35 mg/kg per day 1,2-DAB, but not with 1,3-DAB, developed muscle spasms and progressive weakness, most prominently in hind limbs. Light microscopy revealed swollen axons in spinal anterior horns and proximal ventral roots, and to a lesser extent in dorsal root ganglia of 1,2-DAB-treated animals. Ultrastructural examination of swollen axons revealed clumps of maloriented 10-nm neurofilaments. Sciatic nerves showed clustering of axonal microtubules and other organelles. These findings are qualitatively comparable to those reported in rats treated with 1,2-DAB and represent a suitable phenotype with which to explore molecular mechanisms of proximal, giant neurofilamentous axonopathy using proteomic and genomic technologies.

1,2-diacetylbenzene, the neurotoxic metabolite of a chromogenic aromatic solvent, induces proximal axonopathy.

Several widely used aromatic hydrocarbon solvents reportedly induce blue-green discoloration of tissues and urine in animals and humans. The chomophore has been proposed to result from a ninhydrin-like reaction with amino groups in proteins. The present study examines the neurotoxic property of 1,2-diacetylbenzene (1,2-DAB), the active metabolite of the chromogenic and neurotoxic aromatic solvent 1,2-diethylbenzene. Rats treated with 1,2-DAB, but not with the nonchromogenic isomer 1,3-DAB or with ninhydrin developed blue discoloration of internal organs, including the brain and spinal cord. Only 1,2-DAB induced limb weakness associated with nerve fiber changes, which were most prominent in spinal cord and spinal roots. Changes began with the formation of proximal, neurofilament-filled axonal swellings of the type seen after treatment with 3,4-dimethyl-2,5-hexanedione, a potent derivative of the active metabolite of the neurotoxic aliphatic hydrocarbon solvents n-hexane and methyl n-butyl ketone. These compounds are metabolized to a gamma-diketone that forms pyrroles with target proteins, such as neurofilament proteins. A comparable mechanism is considered for 1,2-DAB, an aromatic gamma-diketone.

Bioactivation of cyanide to cyanate in sulfur amino acid deficiency: relevance to neurological disease in humans subsisting on cassava.

Neurological disorders have been reported from parts of Africa with protein-deficient populations and attributed to cyanide (CN-) exposure from prolonged dietary use of cassava, a cyanophoric plant. Cyanide is normally metabolized to thiocyanate (SCN-) by the sulfur-dependent enzyme rhodanese. However, in protein-deficient subjects where sulfur amino acids (SAA) are low, CN may conceivably be converted to cyanate (OCN-), which is known to cause neurodegenerative disease in humans and animals. This study investigates the fate of potassium cyanide administered orally to rats maintained for up to 4 weeks on either a balanced diet (BD) or a diet lacking the SAAs, L-cystine and L-methionine. In both groups, there was a time-dependent increase in plasma cyanate, with exponential OCN- increases in SAA-deficient rats. A strongly positive linear relationship between blood CN- and plasma OCN- concentrations was observed in these animals. These data are consistent with the hypothesis that cyanate is an important mediator of chronic cyanide neurotoxicity during protein-calorie deficiency. The potential role of thiocyanate in cassava-associated konzo is discussed in relationship to the etiology of the comparable pattern of motor-system disease (spastic paraparesis) seen in lathyrism.

Sodium cyanate alters glutathione homeostasis in rodent brain: relationship to neurodegenerative diseases in protein-deficient malnourished populations in Africa.

Sodium cyanate, a neurotoxic chemical in rodents, primates and humans, is implicated in neurodegenerative disorders in protein-deficient populations subsisting in parts of Africa on the cyanogenic plant cassava. The molecular and cellular mechanisms of cyanate neurotoxicity are not understood. This study investigates the effect of sodium cyanate on glutathione (GSH) homeostasis in rodent brain and liver in vitro and in vivo. GSH levels in mouse brain were rapidly, time- and dose-dependently decreased following intraperitoneal administration of 100, 200 or 300 mg/kg sodium cyanate. By contrast, GSH disulfide (GSSG) levels were increased and GSH/GSSG ratios were decreased in a dose-dependent manner in rat brain. Sodium cyanate depleted GSH levels in all regions of mouse brain. Brain glutathione reductase activity was dose-dependently inhibited, while glutathione peroxidase activity was not affected by sodium cyanate. The disruption of GSH homeotasis, as evidenced by reduced tissue GSH/GSSG ratios, likely results from cyanate-induced inhibition of glutathione reductase activity. The results of this study suggest that cyanate neurotoxicity, and perhaps cassava-associated neurodegenerative diseases, are mediated in part by disruption of glutathione homeostasis in neural tissue.

Dietary deficiency of cystine and methionine in rats alters thiol homeostasis required for cyanide detoxification.

Nutritional status is an important factor in modulating the metabolic fate of xenobiotics. Sulfur amino acid (SAA) deficiency has been proposed as a risk factor for human neurological diseases among protein-poor populations subsisting on the cyanophoric plant cassava. Female Sprague-Dawley rats were used to develop and define a model of SAA deficiency for use in future studies examining cassava-related neurotoxicity. Rats were kept in metabolic cages for 7-21 d and fed a balanced diet (BD) of known composition or a comparable diet selectively deficient in methionine and cystine (SAA-free diet). Animals fed the SAA-free diet failed to thrive, lost body weight, excreted porphyrinic materials, and showed a steep and persistent reduction of urinary inorganic sulfate. In contrast, animals on the BD gained body weight and maintained baseline output of urinary inorganic sulfate. Urinary thiocyanate excretion did not differ between groups, but plasma thiocyanate concentrations reached double that in SAA-deficient rats. Increased plasma thiocyanate suggests mobilization of sulfur amino acids from endogenous sources. Liver glutathione and blood cyanide concentrations were similar in animals on the BD and the SAA-deficient diet. In summary, a diet free of methionine and cystine results in increased retention of inorganic sulfur as thiocyanate and a near absence of inorganic sulfur excretion in urine.

beta-Cyano-L-alanine toxicity: evidence for the involvement of an excitotoxic mechanism.

The legume Vicia sativa (common vetch) harbors the neurotoxic nonprotein amino acid beta-cyano-L-alanine (BCLA) and its gamma-glutamyl derivative. BCLA elicits hyperexcitability, convulsions, and rigidity in chicks and rats after oral or intraperitoneal administration, but the mechanism of its action is unknown. The effect of different concentrations of BCLA (0.075-10.0 mM) has been investigated in an organotypic tissue culture system. BCLA concentrations of 0.075 and 0.60 mM had no effect, even up to 6 hr. No changes were observed in cultures treated with 1 mM BCLA for 4 hr. BCLA (2.0-10.0 mM) induces concentration-dependent changes in the explants. The explants display neurona vacuolation, chromatin, clumping, and dense shrunken cells, a pathological response generally seen with excitotoxin. MK-801 (35 microM), which blocks the open ion channel associated with the N-methyl-D-aspartate (NMDA) class of glutamate receptors, attenuates the neurotoxic property of BCLA, while the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (10-20 microM), provides no significant protection. Treatment of isolated mouse brain mitochondria with up to 5 mM BCLA had no inhibitory effect on the activity of NADH dehydrogenase (complex I) or cytochrome or oxidase (complex IV), a cyanide-sensitive enzyme. These results suggest that the neurotoxicity of BCLA (or derivative) is mediated directly or indirectly through NMDA receptors.

Action of beta-N-oxalylamino-L-alanine on mouse brain NADH-dehydrogenase activity.

beta-N-Oxalylamino-L-alanine (L-BOAA), a non-protein neuroexcitatory amino acid present in the seeds of Lathyrus sativus (chickling or grass pea), is known to produce its neurotoxic effects by overstimulation of non-N-methyl-D-aspartate receptors, especially alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, at micromolar concentrations. It has recently been reported that L-BOAA selectively inhibits mitochondrial enzyme NADH-dehydrogenase (NADH-DH) in brain slices at subpicomolar concentrations. The present study finds that up to 4 mM concentrations of pure L-BOAA fail to inhibit NADH-DH activity in mouse brain homogenate and isolated brain mitochondria. Two known inhibitors (rotenone and 1-methyl-4-phenylpyridinium ion, MPP+) of this mitochondrial enzyme produced significant inhibition under identical conditions. NADH-DH inhibition was also not observed in the homogenate or mitochondria from the brains of animals systemically treated with convulsive doses of L-BOAA. Some inhibition (20-37%) of NADH-DH activity was observed in mouse brain slices incubated with 100-1,000 microM concentrations of L-BOAA for 1 h at 37 degrees C in an atmosphere of 95% O2 and 5% CO2, but the inhibition was nonselective, because the activity of another mitchondrial enzyme, succinic dehydrogenase, was similarly inhibited by L-BOAA. These results are in contrast with the report that L-BOAA inhibits mitochondrial NADH-DH selectively at subpicomolar concentrations. We suggest the observed nonselective NADH-DH inhibition in mouse brain slices treated with L-BOAA is caused by neuronal damage through an excitotoxic mechanism.

Attenuation of glyceraldehyde-3-phosphate dehydrogenase activity and ATP levels in rat brain synaptosomes by acrylamide.

The effect of neurotoxin acrylamide (AC) on energy metabolism has been studied in a purified preparation of the synaptosomes. The synaptosomes were prepared by the flotation technique in a discontinuous Ficoll/sucrose gradient. The purity of the synaptosomes was checked by electron microscopy and by assaying the activity of marker enzymes. By these criterias, free mitochondrial contamination in the synaptosomes was found to be > 2%. Incubation of the synaptosomes with different concentrations of AC (2.5, 5.0, and 10mM) produced a concentration-dependent inhibition (15, 35, and 60%, respectively) of glyceraldehyde-3-phosphate dehydrogenase activity. Acrylamide also produced a time-dependent decrease of ATP concentrations in the synaptosomes; about 25% loss of ATP was seen within 1h, while about 60% ATP was lost after 120 min incubation with 10 mM AC. The effect of known inhibitors of glycolysis-iodoacetic acid (IAA), and of oxidative phophorylation-rotenone and antimycin A, was also studied on ATP synthesis by the synaptosomes. IAA was found to be the most potent inhibitor of ATP synthesis, while both rotenone and antimycin A were equally effective in blocking ATP synthesis in the synaptosomes. These studies show that the synaptosome might be used as a suitable in vitro model to study the effect of neurotoxin such as AC on neuronal energy metabolism.

Acrylamide alters neurofilament protein gene expression in rat brain.

Acrylamide, a prototype neurotoxin, alters neurofilament protein (NF) gene expression in rat brain. Levels of mRNA coding for neurofilament protein subunits NF-L, NF-M, and NF-H have been determined by Northern blot analysis using 32P-labeled cDNA probes. Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls. The expression of NF-L or NF-H mRNA is not affected by acrylamide. In contrast, chronic treatment with acrylamide [0.03% (w/v) in drinking water for 4 weeks] induces a modest but significant increase (approximately 22%) in NF-L mRNA compared to controls. Levels of NF-M, and NF-H mRNA are not altered by acrylamide treatment. The expression of beta-actin mRNA, an ubiquitous protein, is not affected by either treatment regimen of acrylamide. The results of this study show that acrylamide increases the expression of mRNA for NF protein subunits in rat brain. The increase of specific mRNA for NF subunits depends on the dose, duration and route of acrylamide administration.

Acrylamide induces immediate-early gene expression in rat brain.

Northern blot analysis was used to study the effects of acrylamide, a potent neurotoxin, on the induction of c-fos and c-jun mRNA in rat brain. Male Sprague-Dawley rats (10-12 weeks old) treated with acrylamide as a single dose (100 mg/kg, i.p.) or via drinking water (0.03% w/v) for 4 weeks, were used to study acute and chronic effects on immediate-early gene expression, respectively. Acute administration of acrylamide caused a statistically significant increase in the expression of c-fos (approx. 37%) and c-jun (approx. 17%) mRNA in rat brain. By contrast, the level of c-fos mRNA in chronic acrylamide treatment was not altered significantly, but the expression of c-jun mRNA was increased almost 100% as compared to control. These data show that the neurotoxin acrylamide induces immediate-early gene expression in the brain. The effects appear to be related to the route of administration, dose and duration of acrylamide treatment.

Effect of acrylamide on the distribution of microtubule-associated proteins (MAP1 and MAP2) in selected regions of rat brain.

The effect of acrylamide treatment on the immunocytochemical localization of microtubule-associated proteins (MAP1 and MAP2) was studied in different brain regions (cerebellum, cerebral cortex, and hippocampus) of adult rats. Animals were treated with acrylamide (estimated mean dose: 15 mg/kg/d) orally for 2 wk when they showed slight hindlimb weakness. Immunoreactivity for MAP1 and MAP2 was detected in tissue sections with monoclonal antibodies according to the Sternberger's peroxidase-antiperoxidase technique. Intense MAP1 immunoreactivity was observed in neuronal perikarya and dendrites, with faint staining in axons. By contrast, MAP2 immunostaining was selectively observed in dendrites and neuronal perikarya. Treatment of animals with acrylamide reduced immunoreactivity for both MAP1 and MAP2 in hippocampus and cerebellum, with relatively little change in cerebral cortex. Loss of MAPs immunoreactivity in affected brain areas likely proceeded from dendrite to perikaryon. The results of this study indicate that hippocampal compromise is part of the neurotoxic picture associated with rodent exposure to acrylamide.

Acrylamide-induced depletion of microtubule-associated proteins (MAP1 and MAP2) in the rat extrapyramidal system.

Acrylamide, an occupational neurotoxicant, reduced MAP1 and MAP2 distribution in different regions of rat brain. Different components of the extrapyramidal system (caudate-putamen, globus pallidus, substantia nigra and red nucleus) revealed differential distribution of MAP1 and MAP2 in acrylamide-treated animals. Rats were treated with acrylamide (estimated mean dose: 15 mg/kg/day) for 2 weeks and MAP1 and MAP2 were localized according to Sternberger's peroxidase-anti-peroxidase technique. MAP1 labelled neuronal perikarya and dendrites almost with a similar intensity, but MAP2 immunostaining was more intense in dendrites than neuronal perikarya. Acrylamide caused a near-total loss of MAP1 and MAP2 immunoreactivity in caudate-putamen. Other components of the extrapyramidal system were relatively less affected by acrylamide. These results indicate that caudate-putamen is more susceptible to the action of acrylamide than other components of the extrapyramidal system studied. The depletion of MAP1 and MAP2 immunoreactivity by acrylamide appears to be an early biochemical event preceding peripheral neuropathy. The loss of MAPs immunoreactivity occurs first in dendrites and proceeds toward the perikarya. This study indicates that acrylamide not only causes axonal damage but may also induce dendritic degeneration.

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve.

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of 125I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8 +/- 0.4 mm/h) than in mouse sciatic nerves (5.4 +/- 0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0-2.5 h) with a reversal to normal rate 3-5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.

Acrylamide impairs fast and slow axonal transport in rat optic system.

Effects of single and repeated doses of acrylamide on fast and slow axonal transport of radio labeled proteins following the injection of L-[4,5-3H] leucine have been studied in the optic system of male Sprague-Dawley rats. A single dose of acrylamide (100 mg/kg) had no effect, but higher concentrations (200-300 mg/kg) altered the distribution of fast axonally transported materials in optic nerves and optic tracts. Repeated doses of acrylamide (30 mg/kg/day, 5 days per week for 4 weeks) produced degeneration of tibial nerves but spared optic nerves and optic tracts. Fast axonal transport rate in optic axons was reduced by 50% (reduced to 4 mm/h from 8 mm/h) in acrylamide treated animals. Acrylamide also slowed the velocity of slow axonal transport of labeled proteins in optic axons to 1.0 mm per day from 1.3 mm per day. Since acrylamide impaired the rate of both fast and slow axonal transport in the absence of overt morphological damage, it can be concluded that deficit in axonal transport is an important factor in the pathogenesis of axonal degeneration in acrylamide neuropathy.

Determination of polyamines by precolumn derivatization with 9-fluorenylmethyl chloroformate and reverse-phase high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method for the determination of picomole levels of polyamines (putrescine, spermidine, and spermine) is described. Amino groups in polyamines react with 9-fluorenylmethyl chloroformate (FMOC) to form stable and highly fluorescent derivatives which can be separated and quantitatively estimated by HPLC in about 12 min. The mean relative elution times (n = 14) for putrescine, spermidine and spermine are 4.21 +/- 0.02, 10.09 +/- 0.02 and 11.19 +/- 0.04 min, respectively. The method has been applied to determine polyamine concentration in rat dorsal root ganglia (DRG) without interference with endogenous amino acids. Polyamine content of individual rat DRG has been calculated and the values are as follows: putrescine, 36.8 +/- 2.01, spermidine, 1652 +/- 131.0 and spermine 388.5 +/- 38.4 pmol/DRG. Information on polyamine concentrations in DRG may be useful in understanding the mechanism of action of toxic chemicals on nervous system.

Effect of exogenous pyruvate on acrylamide neuropathy in rats.

The protective effect of exogenous sodium pyruvate on the distal-proximal progression of experimental acrylamide neuropathy in rats was examined. Incorporation of 2% (w/w) sodium pyruvate powder in the diet of rats receiving subcutaneous injections of an aqueous solution of acrylamide (35 mg/kg/day, 5 days/week) retarded the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy. Pyruvate supplementation did not alter hexobarbital sleep time or zoxazolamine paralysis time, two in vivo measures of microsomal mixed-function oxidase activity, and the disposition of radioactivity in plasma or sciatic nerve following subcutaneous injection of [14C]acrylamide. Although acrylamide can interfere with energy metabolism at a variety of sites where pyruvate can rescue neurons (axons), the data of this study are consistent with our earlier hypothesis that acrylamide neuropathy may be associated with a glycolytic deficit. The exact site of pyruvate protection is unknown. Exogenous pyruvate is perhaps utilized by axons to circumvent toxin-induced glycolytic inhibition and provide chemical energy for fast axonal transport.

Axotomy-induced ornithine decarboxylase activity in the mouse dorsal root ganglion is inhibited by the vinca alkaloids.

Vinca alkaloids were used to study the role of retrograde axon transport (RT) in activating neuron perikaryal repair response to nerve transection. Mouse lumbar dorsal root ganglia (DRG) (L4-L6) were excised 48 hours after unilateral transection of the sciatic nerve and ornithine decarboxylase (ODC) activity determined. ODC activity in DRG ipsilateral to nerve transection was increased 10-20 fold over contralateral values. Typical ODC activities in ipsilateral and contralateral DRG samples were 6.18 +/- 1.4 and 0.31 +/- 0.09 pmol 14CO2 released/h/3DRG, respectively. Systemic administration of single doses of either vincristine (1 mg/kg) or vinblastine (5 mg/kg) immediately prior to axotomy attenuated ODC induction in ipsilateral DRG by 39% and 47%, respectively. A direct inhibition of ODC activity in the DRG appears unlikely since only high concentrations of vinblastine (0.5-1.0 mM) were able to inhibit ODC activity in vitro. We suggest vinca alkaloids inhibit ODC induction as a consequence of disrupting retrograde axonal transport. Interruption of this intracellular communication mechanism may be etiologically linked to the the distal axon degeneration which follows repetitive exposure to vinca alkaloids and other agents that induce toxic axonal neuropathy.

Effect of hexacarbons on selected lipids in developing rat brain and peripheral nerves.

The effects of neurotoxic solvents, i.e. 2,5-hexanedione (2,5-HD), 2,5-hexanediol (2,5-HDiol) and the non-neurotoxic solvent, 2,4-hexanedione (2,4-HD) (500 mg/kg body wt./day, i.p.), have been studied on the lipid composition of brain and sciatic nerves in weanling rats. Five-day-old rats were administered a solvent daily for 21 days. Clinical signs of peripheral neuropathy appeared in 2,5-HD and 2,5-HDiol treated groups. Absolute weights of brain, spleen, thymus significantly decreased with 2,5-HD. Cholesterol content in whole brain homogenates and myelin was significantly reduced with 2,5-HD and 2,5-HDiol treatment. There was also a significant reduction in ubiquinone content of brain with 2,5-HD and 2,5-HDiol treatment. On exposure to neurotoxic chemicals to weanling rats, significant alteration in lipid profile was observed in the brain, which may be one of the key factors in the development of neuropathy.