Richard D. Howland

The etiology of toxic peripheral neuropathies: In vitro effects of acrylamide and 2,5-hexanedione on brain enolase and other glycolytic enzymes

The in vitro effects of the neurotoxic compounds, acrylamide and 2,5-hexanedione, on several glycolytic enzymes including enolase, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactic dehydrogenase (LDH) were studied in rat brain. A differential sensitivity of the enzymes to the inhibitory effects of the neurotoxins was observed. The order of increasing sensitivity to 2,5-hexanedione was enolase -- GAPDH -- PFK and to acrylamide the order was PFK -- enolase -- GAPDH. Neither neurotoxin inhibited LDH. The inhibition of enolase by acrylamide exhibited a mixed type pattern in double reciprocal plots. The inhibition could be completely reversed by dialysis indicating that it did not involve covalent bond formation. In the presence of dithiothreitol (DTT) or glutathione the inhibition of enolase by either acrylamide or 2,5-hexanedione was potentiated. Activity of enolase inhibited by both acrylamide and DTT could not be restored to pre-inhibition rates following dialysis indicating that an irreversible interaction between acrylamide and enolase had taken place. The results suggest that neurotoxic compounds which produce distal axonopathies have a common pattern of attack on glycolytic enzymes and that interruption of glycolysis is the underlying biochemical basis for both the physiological and morphological damage caused by these compounds.

The etiology of acrylamide neuropathy: Enolase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase activities in peripheral nerve, spinal cord, brain, and skeletal muscle of acrylamide-intoxicated cats

Previous studies have shown that the neurotoxin, acrylamide, inhibits several glycolytic enzymes when tested in vitro and that the activity of neuron-specific enolase (NSE) is decreased in rats treated with neurotoxic doses of acrylamide. To determine whether the effect on NSE was species specific and whether glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PFK) activities were decreased in vivo, cats were treated with either 15 or 30 mg/kg/day of acrylamide for 10 days. Twenty-four hours after the last injection, samples of sciatic nerve, brain, spinal cord, and skeletal muscle were assayed for total enolase, NSE, GAPDH, and PFK activities. The low and high doses of acrylamide resulted in the following respective decreases in NSE activity: 62.1 and 69.7% in the distal peripheral nerve and 37.1 and 45.4% in brain. GAPDH activity was similarly depressed in brain and both proximal and distal portions of the sciatic nerve. Additionally, all other tissues showed significant reductions in GAPDH. Neither total enolase activity of peripheral nerve, spinal cord, and skeletal muscle nor PFK activity in these tissues and in brain was decreased. In contrast, PFK in gastrocnemius muscle was increased 700% by the high dose of acrylamide, which may represent a compensatory mechanism. These data support the hypothesis that the mechanism of the toxic action of acrylamide involves inhibition of glycolysis at two enzymatic sites and that the selectivity exhibited by the toxic agent resides in its ability to inhibit a neuron-specific isoenzyme.

The etiology of acrylamide neuropathy: Possible involvement of neuron specific enolase

The effect of monomeric acrylamide, a potent neurotoxic agent, on total and neuron specific enolase activity was studied in vitro and in vivo. Acrylamide (10 mM) completely inhibited total enolase activity of rat brain soluble fractions. The I50 concentration was 3.7 mM. In rats chronically treated with acrylamide (550 mg/kg total) and exhibiting marked symptoms of neurotoxicity, neuron specific enolase activity was not detectable in sciatic nerves and was only 60% of control activity in brain. Total enolase activity in both central and peripheral nervous tissues was unchanged from control. The results suggest that inhibition of neuron specific enolase may be an important factor in the development of acrylamide neuropathy by interfering with glycolysis in neuronal tissue.

Altered phosphorylation of rat neuronal cytoskeletal proteins in acrylamide induced neuropathy

The activity of protein kinase has been assayed in neurofilament preparations from spinal cords of rats treated with acrylamide. Animals received 50 mg/kg, i.p., of acrylamide per day for a total of 5 or 10 days; these doses produce mild and marked symptoms of neurological damage, respectively. Incorporation of phosphate into proteins was determined using [gamma-32P]ATP followed by SDS-PAGE. Total phosphorylation of neurofilament preparations was significantly increased only in the animals treated with the 500 mg/kg cumulative dose of acrylamide. Phosphorylation of the 200 and 155 kdalton subunits of the neurofilaments was increased by 20-40% in the acrylamide treated groups. The phosphorylation of the 70 kdalton neurofilament subunit was unchanged in the 250 mg/kg group and was significantly decreased in the 500 mg/kg group. Phosphorylation of other protein bands was not altered. These results suggest a mechanism by which acrylamide might produce axonal neurofilamentous accumulations.

DFP mononeuropathy: Evidence for a peripheral site of initiation

The foci at which neurotoxic organophosphorous compounds induce axonopathies have been thought to be the neural perikarya, with subsequent retrograde axonal degeneration resulting from interference with metabolic function in the soma 5. However, recent studies raise the possibility that the organophosphorous 3,4 and other neurotoxic agentsT,9,10 impact directly on the axon and the perikarya are not involved in the etiology of the axonopathy. A major difficulty in determining sites of initiation of axonal degeneration results from systemic administration of the toxic agent. A technique to circumvent this difficulty is to produce a localized, mononeuropathy by restricting the distribution of the neurotoxic agent. Diisopropylfluorophosphate (DFP) injection into one femoral artery of cats results in a mononeuropathy in the injected hindlimb, confirmed by physiologicaP,2, 8 and morphological2, 6 criteria. However, these studies do not definitely rule out perikaryal involvement in the neuropathy since the distribution of DFP following intraarterial injection is unknown. The present studies confirm that DFP is largely restricted to the injected leg, with relatively small amounts reaching the contralateral limb or the spinal cord. Ten adult mongrel cats were anesthetized with sodium pentobarbital (25 mg/kg i.v.), the left femoral artery exposed, and DFP (K and K Labs) (2 mg/kg) injected by means of a 30-gauge needle just below the level of the superficial epigastric artery. [3H]DFP (spec. act. 3.4 Ci/mmol, Amersham and Searle) was added to the injection solution to achieve approximately 20 × l0 G cpm/kg body weight. Concentrations of solutions were adjusted so that the desired dosage was injected in a volume of 0.1 ml/kg body weight. An equal volume of saline was injected into the contralateral femoral artery. All solutions were freshly prepared just prior to injection.

Peripheral nerve phospholipids in acrylamide neuropathy

Treatment of cats with acrylamide, either 7.5 or 15 mg/kg IM, once a day for 10 days, resulted in increases of 31 and 47% in the phospholipid content of sciatic nerve, respectively, from a control level of 41.1±2.7 mg/kg wet weight. Determination of the distribution of individual phospholipids indicated no significant differences between control cats and those receiving a cumulative dose of 150 mg/kg acrylamide. In a separate experiment, cats were treated with the 150 mg/kg dose of acrylamide and the sciatic nerve was divided into proximal and distal portions at the level of the triceps surae nerve. Significant increases in phospholipid content were observed in both the proximal and distal portions of peripheral nerve of the acrylamide-intoxicated cats. This effect was present even when the phospholipid content was expressed in terms of total protein, dry weight or total lipid. Total weight of nerve segments, however, was significantly decreased in the neuropathic animals. The data are consistent with a focal degeneration of axons with relative sparing of phospholipids.

Neuron-specific enolase: investigation on its possible retrogade axonal transport

Abstract The possible rapid retrograde axonal transport of neuron-specific enolase was investigated employing a sensitive radioimmunoassay. The right sciatic nerve of rats was ligated and 6 and 24 h later the nerve was cut in two 5.0 mm segments immediately proximal and distal to the ligature. Similar were taken from the contralateral nerve as a control. No increase in the amount of neuron-specific enolase in the right-distal segment was observed indicating that the protein is not a component of retrograde axonal transport.