Daniel M. Lapadula

Cross‐Linking of Neurofilament Proteins of Rat Spinal Cord In Vivo After Administration of 2,5‐Hexanedione

The aliphatic hexacarbons n‐hexane, methyl‐nbutyl ketone, and 2,5‐hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10‐nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5‐hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate‐polyacrylamide gel electro phoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti‐70K, anti‐160K, and anti‐210K neurofilament antibodies revealed many crosslinked peptides. The 138K band cross‐reacted with the anti‐160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross‐link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high‐molecular‐weight peptides immuno reactive with all three neurofilament protein antibodies. In addition to cross‐linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5‐hexanedione‐induced cross‐linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.

In vitro binding of [14C]acrylamide to neurofilament and microtubule proteins of rats

Acrylamide produces a dying back type of neuropathy in which there is an accumulation of neurofilaments in the axons. The in vitro binding of [14C]acrylamide to neurofilament and microtubule proteins obtained from rat spinal cord and brain was investigated. The relative binding to the high and middle molecular weight neurofilament was greater than to the low molecular weight neurofilament, while the rate of binding to MAP-1 (microtubule associated protein-1) and -2 was much greater than to tubulin. The binding rate to a 53 kDa protein which co-purified with the neurofilaments was between those of the middle and high molecular weight neurofilaments while the lowest rate of binding was to glial fibrillary acidic protein. These data indicate that there is a direct binding of acrylamide to cytoskeletal proteins.

Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy

The present studies were carried out to investigate the comparative roles of protein cross-linking and alteration in protein phosphorylation in the accumulation of neurofilaments due to aliphatic hexacarbons. In these studies, rats were given 2,5-hexanedione (0, 0.1, 0.25 and 1.0%) for 70 days in their drinking water. In a separate study of in vitro protein phosphorylation rats were given 1% 2,5-hexanedione for 14 days in their drinking water. Spinal cord neurofilaments were isolated and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using anti-neurofilament antibodies, radioimmunoassays (RIAs) of phosphorylated epitopes on neurofilament proteins and protein phosphorylation. Protein cross-linking of neurofilaments was found in all animals treated with 2,5-hexanedione including the lowest dose (0.1%) which did not produce clinical signs of intoxication. Protein phosphorylation of neurofilament proteins, as well as MAP-2 was significantly decreased upon treatment. Protein staining revealed a decreased amount of neurofilament protein and immunoblotting demonstrated neurofilament protein cross-linking in these animals. Protein staining of glial fibrillary acidic protein (GFAP) was unaltered by this treatment. RIAs of phosphorylated and non-phosphorylated epitopes of neurofilament proteins indicated that in vivo phosphorylation of these proteins was also decreased. Two-dimensional gel electrophoresis indicated a shift of the neurofilament proteins to a basic pI, indicating a dephosphorylation of neurofilament proteins. Cross-linked neurofilament proteins also exhibited a pI which was more basic than any of the individual neurofilament proteins. This report demonstrates differential effects of 2,5-hexanedione on neurofilament proteins and indicates that several mechanisms may be responsible for their accumulation.

Neurotoxicity of glycidamide, an acrylamide metabolite, following intraperitoneal injections in rats

Acrylamide (2-propenamide) monomer produces central-peripheral distal axonopathy in humans and some animal species. Its neurotoxicity is characterized by abnormal sensation, decreased motor strength, and ataxia. Acrylamide forms adducts with glutathione, proteins, and DNA. Recent studies demonstrated that acrylamide is metabolized to its epoxide, glycidamide (2,3-epoxy-1-propanamide). We studied the neurotoxicity potential of glycidamide in male Sprague-Dawley rats. Animals (groups of 6) were injected ip daily with either aqueous acrylamide or glycidamide at an acrylamide-equivalent dose of 50 mg/kg (0.70 mmol/kg). Both treatments resulted initially in the rats circling, which was followed by the onset of ataxia at 7-9 d and hindlimb paralysis at 12-14 d. Treated animals showed muscle wasting. At termination, acrylamide- and glycidamide-treated rats weighed 105% and 86% of initial weight, respectively, compared to 145% for controls. Animals were anesthetized and perfused with 10% neutral phosphate-buffered formalin 12 or 14 d after beginning of treatment. Both treatment groups exhibited similar neuropathologic changes in the central and peripheral nervous systems. More severe lesions were produced by glycidamide. A marked increase in the number of affected Purkinje cells in the cerebellum, which exhibited changes ranging from pyknosis to cell death, were present. The brainstem exhibited axonal degeneration with chromatolytic necrosis in midbrain medial and lateral reticular nuclei. The spinal cord was characterized by spongy form changes with vacuoles of different sizes in various levels. These results suggest that glycidamide is an active neurotoxic metabolite of acrylamide.

Cystoskeletal proteins as targets for organophosphorus compound and aliphatic hexacarbon-induced neurotoxicity

Concurrent exposures to organophosphorus insecticide leptophos and the industrial solvents n-hexane and toluene were implicated in causing an outbreak of neuropathy in workers. Although both leptophos and n-hexane produce central-peripheral distal axonopathy, the morphology and distribution of neuropathic lesions are distinct, reflecting different modes of action. The molecular mechanisms of organophosphorus compound-induced delayed neurotoxicity (OPIDN) and aliphatic hexacarbon-induced neurotoxicity have been investigated utilizing various biochemical techniques, (i.e. one- and two-dimensional gel electrophoresis, immunoblotting, peptide mapping). Oral administration of tri-o-cresyl phosphate (TOCP) produced delayed neurotoxicity and increased in vitro Ca2+ and calmodulin-dependent kinase protein phosphorylation of cytoskeletal proteins in brain, spinal cord, and sciatic nerve of chickens. This enhanced protein phosphorylation correlated well with the following characteristics of OPIDN: test chemical, whether an OPIDN-producing or not; dose-dependence and time course of the effect; and the animal sex sensitivity, age selectivity, and species susceptibility. The proteins that showed an increased phosphorylation were identified to be; alpha- and beta-tubulin, microtubule-associated protein-2 (MAP-2), and the 3 neurofilament proteins 70 kDa, 160 kDa, and 210 kDa. Further studies suggested that the increased protein phosphorylation is not related to an effect on protein phosphatase or ATPase activity, but rather to altered Ca2+-calmodulin kinase II activity. Aliphatic hexacarbon-induced neurotoxicity is characterized by an accumulation of 10 nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. Treatment of rats with 2,5-hexanedione, the active neurotoxic metabolite of n-hexane, produced protein crosslinking in a dose-dependent manner. This treatment also decreased protein phosphorylation of neurofilament proteins as well as MAP-2. These studies demonstrate the involvement of cytoskeletal proteins in the molecular pathogenesis of chemical-induced neurotoxicity.

Reproductive tract lesions resulting from subchronic administration (63 days) of tri-o-cresyl phosphate in male rats

An initial dose-range pilot study where animals were gavaged with between 100 and 1600 mg tri-o-cresyl phosphate (TOCP)/kg/day for 14 days resulted in decreased epididymal sperm density and disruption of the seminiferous epithelium in 100% of treated animals. A subchronic 63-day study (reflecting the 49-day length of the rat seminiferous epithelium cycle plus the 14-day transit time of spermatids through the epididymis was initiated. Dose-dependent (10 to 100 mg TOCP/kg/day) decreases in cauda epididymal sperm motility and density, testicular enzyme activities, and alterations in sperm morphology were observed. Concurrent pair-fed controls (matched to the highest dose group, 100 mg TOCP/kg/day) indicated that weight loss resulting from TOCP administration had minimal contributory effects to the testicular toxicity seen. Plasma alpha-tocopherol acetate (vitamin E) and testosterone concentrations were unaffected. Tri-p-cresyl phosphate (TPCP), the nonneurotoxic structural analog of TOCP, produced no toxic effects, demonstrating the necessity of the ortho-cresol moiety for induction of damage. A minimum effective (threshold) dose for observable testicular toxicity was determined to be 10-25 mg TOCP/kg in this study. These data suggest that TOCP interferes with spermatogenic processes and sperm motility directly and not via an androgenic mechanism or decreased vitamin E availability.

Characterization of delayed neurotoxicity in the mouse following chronic oral administration of tri-o-cresyl phosphate

The sensitivity of the mouse to organophosphorus-induced delayed neurotoxicity (OPIDN) has been investigated. One group of five mice received two single 1000-mg/kg po doses of tri-o-cresyl phosphate (TOCP) at a 21-day interval (on Days 1 and 21 of the study); a second group of five mice was given 225 mg/kg of TOCP daily for 270 days. A third group of five animals served as an untreated control. All animals were killed 270 days after the start of the experiment. Daily po dosing of 225 mg/kg TOCP caused a decrease in body weight gain, muscle wasting, weakness, and ataxia which progressed to severe hindlimb paralysis at termination. On the other hand, po administration of two single 1000-mg/kg doses of TOCP at a 21-day interval produced no observable adverse effects. Brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activity were 35 and 10% of the control, respectively, in daily dosed animals while AChE and NTE in mice receiving two single 1000-mg/kg doses of TOCP were not significantly altered from the control group. Plasma butyrylcholinesterase activity was 12% of the control group in daily dosed animals. Hepatic microsomal enzyme activities of aniline hydroxylase and p-chloro-N-methylaniline demethylase and NADPH-cytochrome P-450 content in daily dosed animals were increased (141 to 161% of the control group) when compared to controls and mice receiving two single 1000-mg/kg doses of TOCP; the latter being not significantly different from each other. Degeneration of the axon and myelin of the spinal cord and sciatic fascicle were observed and were consistent with OPIDN. This study demonstrates that chronic dosing of TOCP produces OPIDN and induces hepatic microsomal enzyme activity in mice. It is concluded that while the mouse is susceptible to OPIDN, it is a less sensitive and a less appropriate test animal for studying this effect when compared to the adult hen.

Changes in in vitro brain and spinal cord protein phosphorylation after a single oral administration of tri-o-cresyl phosphate to hens

Abstract: The effect of a single oral 750 mg/kg dose of tri‐o‐cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP‐treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [γ‐32P]ATP was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol—50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes—50K (622%) and 60K (697%); and spinal cord cytosol—20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.

Light and electron microscopic evidence of tri-o-cresyl phosphate (TOCP)-mediated testicular toxicity in Fischer 344 rats

The onset and development of testicular lesions following tri-o-cresyl phosphate (TOCP) dosing have been documented through light and electron microscopic morphological studies. Male Fischer 344 rats (190-210 g body weight) were administered 150 mg TOCP/kg/day in corn oil for 1, 3, 5, 7, 10, 14, and 21 days. Vehicle-treated rats served as the control group. Sections of formaldehyde- and glutaraldehyde-fixed, methacrylate-embedded testes showed, by Day 5, numerous spermatid heads apparently detached from tails lying at oblique angles near the basement membrane of the seminiferous tubules. Columnar and spherically shaped vacuoles of the epithelium, radiating from the basement membrane to the lumen of the tubules, were also observed. Electron micrographs revealed that these were localized in Sertoli cells. Widespread dilation of Sertoli cell smooth endoplasmic reticulum was also noted. By 7 days of treatment, residual body abnormalities were noted in stage VIII tubules, along with spermatocyte-derived multinucleated giant cells. The lesion progressed with increased vacuolation of the epithelium and numbers of abnormal residual bodies and giant cells, together with spermatid karyorrhexis (Days 10, 14, and 21). There was also an apparent decrease in sperm density/tubule with continued exposure: 90% of the seminiferous tubules were devoid of sperm by Day 14. These morphological results indicate an initial effect of TOCP on Sertoli cells. Spermatogenesis is affected as seen by the decrease in sperm density and increase in necrotic spermatids.

Time course of the tri-o-cresyl phosphate-induced testicular lesion in F-344 rats: Enzymatic, hormonal, and sperm parameter studies

Tri-o-cresyl phosphate (TOCP), a known neurotoxic compound, causes testicular toxicity in both leghorn roosters and Fischer 344 rats. The present study was initiated to follow the onset of the testicular lesion through possible changes in sperm numbers and production, serum hormones, and various enzyme activities. Rats were administered TOCP daily (150 mg/kg) for periods of 3, 7, 10, 14, or 21 days. Vehicle-treated animals served as controls. Sperm motility and sperm number per milligram cauda epididymis were both lower in treated animals by Day 10. Testicular weight to body weight ratio was significantly decreased only in the longest treatment duration animals (21 days). Testicular neurotoxic esterase and nonspecific esterase activities were also inhibited, while beta-glucuronidase activity was not affected. Luteinizing and follicle-stimulating hormone levels were normal, as were both serum and interstitial fluid testosterone concentrations. Sertoli cell fluid secretion, as measured by testis weight increase after efferent duct ligation, showed no significant changes. Other organs (spleen, liver, kidney, pancreas, small intestine, adrenal and pituitary glands) had no overt signs of pathology as observed by light microscopy in animals treated for 21 days. A separate group of animals was treated for 21 days and subsequently examined after 98 days of observation (two cycles of the rat seminiferous epithelium). No recovery of spermatogenesis was seen, indicating that the toxicity was irreversible at the dose used. The effects noted in these studies further define the testicular lesion produced by TOCP and show that 150 mg/kg/day for 21 days produced irreversible testicular toxicity.