Evaluation of Therapeutics for Advanced-Stage Heart Failure and Other Severely-Debilitating or Life-Threatening Diseases.

Severely-debilitating or life-threatening (SDLT) diseases include conditions in which life expectancy is short or quality of life is greatly diminished despite available therapies. As such, the medical context for SDLT diseases is comparable to advanced cancer and the benefit vs. risk assessment and development of SDLT disease therapeutics should be similar to that of advanced cancer therapeutics. A streamlined development approach would allow patients with SDLT conditions earlier access to therapeutics and increase the speed of progression through development. In addition, this will likely increase the SDLT disease therapeutic pipeline, directly benefiting patients and reducing the economic and societal burden of SDLT conditions. Using advanced-stage heart failure (HF) as an example that illustrates the concepts applicable to other SDLT indications, this article proposes a streamlined development paradigm for SDLT disease therapeutics and recommends development of aligned global regulatory guidance.

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.

Protein synthesis inhibition blocks maintenance but not induction of epileptogenesis in hippocampal slice.

We have been examining the role of protein synthesis in the development and maintenance of spontaneous bursting in the rat hippocampal slice. We used stimulus train induced bursting (STIB) as an in vitro model for epileptogenesis, to study the effects of 3 different protein synthesis inhibitors (cycloheximide, anisomycin, puromycin) on the development of bursting. We report here that none of these inhibitors blocked the induction of bursting, suggesting that protein synthesis is not essential for the development of electrically induced bursting. However, when established spontaneous bursting was examined in the presence of cycloheximide, the duration of the bursting phase was markedly reduced, suggesting that the maintenance of spontaneous bursting in the early hours requires ongoing protein synthesis.

Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with tri-ortho-cresyl phosphate (TOCP).

Previous biochemical studies demonstrated a dramatic increase in phosphorylation of cytoskeletal proteins that occurs early in organophosphorus ester-induced delayed neurotoxicity (OPIDN). In this report we present immunohistochemical evidence that there is anomalous aggregation of phosphorylated neurofilaments within central and peripheral axons following organophosphate exposure. The morphology, location, and time of appearance of these aggregations are consistent with the hypothesis that the aberrant phosphorylation of cytoskeletal elements is an antecedent to the focal axonal swelling and degeneration characteristic of OPIDN.

Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of alpha and beta/beta' subunits of molecular weights 46,000 and 55,000/52,000, respectively. The ratio of alpha to beta/beta' subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The Km value of ATP was 19 +/- 1.3 microM, and the K0.5 values of calcium and calmodulin were 4.4 +/- 0.5 and 3.0 +/- 0.5 microM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the alpha-subunit but apparently not of the beta/beta' subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II alpha subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both alpha and beta subunits of hen brain CaM-kinase II cross-reacted with only the alpha subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II in several properties. Immunocross-reactivity suggested that there was similarity in the alpha but not the beta/beta' subunits of the hen brain enzyme and the brain enzyme of other species.

Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins.

Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.

Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells.

Primary cultures of bovine adrenomedullary chromaffin cells were treated with the phosphorus acid ester triphenyl phosphite (TPP), a chemical capable of producing Type II organophosphorus compound-induced delayed neurotoxicity (OPIDN), and the morphological changes were assessed by transmission electron and scanning microscopy. Following a 24-hr incubation with 100 microM TPP nearly all mitochondria were either disrupted or swollen and glycogen buildup within the cytoplasm was evident. The viability of cells treated with TPP and cultured on coverslips for scanning electron microscopy was very low. By scanning electron microscopy, the filopodia of these cells appeared contracted. The surface texture was very irregular and giant globular bodies were evident. Parallel studies were carried out with the cholinergic compound O,O-diethyl 4-nitrophenyl phosphate (paraoxon) and the Type I delayed neurotoxicant O,O-diisopropylphosphorofluoridate (DFP). Transmission and scanning electron microscopy revealed that treatment with these organophosphorus compounds did not produce the ultrastructural effects that were seen with TPP. The morphological data were confirmed biochemically by assessing the viability of the mitochondria via measurement of [3H]adenosine incorporation into ATP. Treatment with 100 microM TPP for 4 or 24 hr caused a marked inhibition (90% relative to controls) of adenosine incorporation. Neither 100 microM paraoxon nor 100 microM DFP had an inhibitory effect on incorporation. The effect of TPP was time-dependent with significant biochemical effects as early as 60 min. In contrast, ultrastructural changes were not seen until 24 hr. Morphologically, the 60-min incubations showed no perturbation in mitochondrial integrity. Our results support a specific effect of the triphenylphosphite, TPP, a Type II OPIDN compound, not a general toxic effect of organophosphorus compounds since the cholinergic agent paraoxon and the Type I delayed neurotoxic compound DFP did nto alter the cells ultrastructurally or compromise the mitochondria biochemically. The apparent target for TPP toxicity is the mitochondria.

Persistent alterations of calmodulin kinase II activity in chickens after an oral dose of tri-o-cresyl phosphate.

Calmodulin kinase II has been found to be involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing organophosphorus compound-induced delayed neurotoxicity (OPIDN). In this report, chickens were given a single oral neurotoxic dose of 750 mg/kg tri-o-cresyl phosphate (TOCP), and killed after 1 or 21 days of treatment. Crude calmodulin kinase II from brain cytosol as well as phosphocellulose-purified microtubules were prepared from control and treated animals. Phosphorylation reactions were started by adding protein into the phosphorylation buffer in the presence of Mg2+, Ca2+, calmodulin or trifluoperazine, and [gamma-32P]ATP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to autoradiography. The extent of the calmodulin kinase II autophosphorylation as well as the Ca2+/calmodulin-dependent phosphorylation of the purified microtubules was investigated. The enzyme activities isolated from control and treated animals were compared. Autophosphorylation of calmodulin kinase II was found to be higher in both 1-day and 21-day TOCP-treated animals than in control animals. The activity of the kinase to phosphorylate exogenous substrates such as tubulin and microtubule-associated protein-2 (MAP-2) was also higher in the treated hens than in the controls. The increased activity of the kinase was noted at day 1 following treatment when no clinical signs were observed and persisted until day 21 when the animals were paralyzed completely. This finding supports the significance of altered calmodulin kinase II in the pathogenesis of OPIDN.

Mechanisms of joint neurotoxicity of n-hexane, methyl isobutyl ketone and O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens.

The joint neurotoxic action of simultaneous exposure to vapors of n-hexane and methyl iso-butyl ketone (MiBK) and dermally applied O-ethyl O-nitrophenyl phenylphosphonothioate (EPN) was studied in groups of five adult hens. Four groups of hens were concurrently exposed to a dermal 2.5 mg/kg of EPN, 1000 ppm of n-hexane and 100, 250, 500 or 1000 ppm of MiBK. Two groups were each exposed to binary mixtures of a dermal dose of 2.5 mg/kg of EPN and 250 ppm of MiBK or 1000 ppm of n-hexane. Another three groups of hens were exposed to either 250 ppm of MiBK, 1000 ppm of n-hexane or a dermal dose of 2.5 mg/kg of EPN. A Group of hens was kept untreated. All hens were terminated after 30 days of treatment. Hens exposed to MiBK or n-hexane vapor did not exhibit any toxicity signs. In contrast, hens treated with EPN alone or in combination with n-hexane and/or MiBK developed acute cholinergic and delayed neurotoxicity signs. Hen brain acetylcholinesterase and neurotoxic esterase activities were inhibited in hens treated concurrently with EPN, n-hexane and MiBK. MiBK alone or in combination with EPN and n-hexane induced liver microsomal cytochrome P-450 content and phenobarbital-inducible cytochrome P-450 enzyme activities. Microsomes from hens treated with EPN, n-hexane, MiBK or mixtures of EPN, n-hexane and MiBK significantly enhanced the biotransformation of EPN to the more neurotoxic oxidation metabolite O-ethyl O-4-nitrophenyl phenylphosphonate.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of cytochrome P450 isozymes by simultaneous inhalation exposure of hens to n-hexane and methyl iso-butyl ketone (MiBK).

Chickens were exposed simultaneously to the industrial hexacarbon solvents n-hexane and methyl iso-butyl ketone (MiBK). n-Hexane has been shown to be neurotoxic in both humans and other vertebrates. While MiBK is not neurotoxic, it has been shown to greatly synergize the clinical appearance of neurotoxicity in animals exposed to both of these solvents. Groups of hens were exposed for 29 days in inhalation chambers to 1000 ppm n-hexane in combination with 10, 100, 250, 500, or 1000 ppm MiBK. Other groups received either 1000 ppm n-hexane, 1000 ppm MiBK, or ambient air and served as controls. A dose-dependent decrease in body weight and an increase in clinical effects were noted for the highest exposure groups (1000 ppm n-hexane combined with 1000, 500 or 250 ppm MiBK). There was an MiBK dose-dependent increase in cytochrome P450 content and benzphetamine N-demethylase activity, but there was no distinct pattern for ethoxyresorufin O-deethylase or cytochrome c reductase activities. Mixed-function oxidase levels and activities (cytochrome P450 content and benzphetamine N-demethylase) were elevated significantly (P less than 0.05) over controls even in the lowest MiBK group (10 ppm), although there were no clinical signs of neurotoxicity. Four different isozymes of cytochrome P450 were measured immunologically. There was a dose-dependent increase in three of the isozymes, two of which were phenobarbital inducible and one of which was induced by beta-napthoflavone. Quantitatively, the largest increase was in the PB-A isozyme, a phenobarbital-inducible isozyme which accounted for approximately 70% of the cytochrome P450 present in animals treated with MiBK. The results suggest that MiBK selectively induces cytochrome P450 isozymes leading to the metabolic activation of the weak neurotoxicant n-hexane to the potent neurotoxicant 2,5-hexanedione (2,5-HD).

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.

In vivo binding of [(14)C]acrylamide to proteins in the mouse nervous system.

The labeling of mouse brain and spinal cord proteins in vivo following the administration of 200 ?g/kg [(14)C]acrylamide (25 mCi/kg, i.p.) was measured by polyacrylamide gel electrophoresis and autoradiography. Several cytoskeletal proteins had the highest specific activity. These included the medium (130 kDa) and high (180 kDa) molecular weight neurofilament proteins and unknown proteins with molecular weights of 31, 33 and 57 kDa. About 0.05% of the neurofilament proteins were labeled with the dose employed. Although accurate assessments of the stoichiometry for acrylamide binding to 31, 33 and 57 kDa bands could not be made due to the low concentrations of these proteins, it is estimated that over 0.1% of these proteins bound acrylamide. There were significant amounts of acrylamide bound to proteins seven days after exposure, with labeling ranging from 15 to 60% of the levels observed one day after exposure. These results show that acrylamide binds to several major cytoskeletal proteins present in the mouse nervous system. The formation of these adducts may play a role in the neurotoxicity of acrylamide.

Purification and characterization of cytochrome P450 isozymes from beta-naphthoflavone-induced adult hen liver.

Cytochromes P450 beta NF-A, beta NF-B, and beta NF-C were purified from beta-naphthoflavone-treated adult hens. Cytochrome P450 beta NF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 beta NF-A1 and beta NF-A2 for property comparison. The cytochromes beta NF-A1 and beta NF-A2 were induced by both phenobarbital and beta-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH2-terminal amino acid sequence. However, P450 PB-A differed from beta NF-A1/beta NF-A2 in peptide pattern after partial proteolysis by alpha-chymotrypsin and Staphylococcus aureus V8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from beta NF-A1/beta NF-A2, in that its antibodies cross-reacted with P-450 of normal, PB-, and beta-NF-induced rabbit liver microsomes. The cytochromes beta NF-B and beta NF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH2-terminal amino acid sequence. The most notable difference between beta NF-B and beta NF-C was that the anti-beta NF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by beta-NF-induced microsomes. These activities increased 40- to 50-fold in beta-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of beta NF-B and beta NF-C were different from those of mammalian and other nonmammalian species.

Effects of diisopropyl phosphorofluoridate (DFP) on CA3 and CA1 responses in rat hippocampus.

Diisopropyl phosphorofluoridate (DFP), an insecticide, is a potent anticholinesterase that binds essentially irreversibly to acetylcholinesterase, resulting in severe, acute neurologic pathology, and less severe, but longer-lasting, delayed neuropathy. We report here on the short-term effects of bath-applied DFP on extracellularly recorded responses from CA3 and CA1 of rat hippocampus. Exposure to 10 microM DFP evokes low amplitude, spontaneous bursts in CA3 generally within 10 minutes, and the bursting does not reverse with washing. The CA1 neuronal population usually bursts synchronously with CA3, but the population events are of low amplitude and sometimes not detectable, implying a differential sensitivity to DFP. These effects were partially blocked by the muscarinic antagonist atropine, while the cholinergic antagonist gallamine had little effect. Also, the reversible anticholinesterase physostigmine could, within temporal limits, protect slices from DFP's effects, implicating the cholinergic system as the probable mediator in the first stages of DFP-induced epileptogenesis.

Assessment of the delayed neurotoxicity of tributyl phosphate, tributoxyethyl phosphate, and dibutylphenyl phosphate.

There industrial organophosphorus compounds were tested for their ability to cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the adult hen. The compounds tested were tributyl phosphate (TBP), tributoxyethyl phosphate (TBEP), and dibutylphenyl phosphate (DBPP). The acute oral LD50 of TBP and DBPP were estimated to be 1,863 and 1,500 mg/kg, respectively, and the dose equal to the LD50 was used as a test dose. The acute oral LD50 of TBEP was greater than 5,000 mg/kg and 5,000 mg/kg was used as a test dose. An oral dose of 750 mg tri-o-cresyl phosphate (TOCP) was used as a positive control. For the acute delayed neurotoxicity test, hens were given two test doses of the test materials 21 days apart and killed 21 days after the second dose. None of the hens given TBP, TBEP, or DBPP exhibited nerve damage or clinical signs which distinguished them from untreated control animals. A single dose of TOCP resulted in paralysis and a histopathological profile typical of a distal neuropathy. For the assay of the inhibition of esterases, hens were killed 24 hours after a single dose equal to the greater of either the LD50 or 5000 mg/kg. TOCP administration resulted in over 90% inhibition of brain neurotoxic esterase (NTE), but none of the other three compounds inhibited NTE to an extent (greater than 70%) which would be expected to result in OPIDN. Administration of TOCP, TBEP, or DBPP resulted in approximately a 70% decrease in plasma butyrylcholinesterase (BuChE) activity. TBP caused a 2-3 fold increase in BuChE activity. TBEP administration resulted in about 45% inhibition of acetycholinesterase (AChE) in brain. These results indicate that TBP, TBEP, and DBPP are all unlikely to cause OPIDN with any single sublethal dose.

Absence of delayed neurotoxicity and increased plasma butyrylcholinesterase activity in triallate-treated hens.

Triallate (S-2,3,3-trichloroallyl diisopropylthiocarbamate) was tested for the potential to produce delayed neurotoxicity. Hens were given single oral doses ranging from 312.5 to 2500 mg/kg of triallate, 750 mg/kg tri-o-cresyl phosphate (TOCP), or empty gelatin capsules on Days 1 and 21 and were killed on Day 42. In a second experiment, animals were administered daily oral doses of 25-300 mg/kg triallate or 10 mg/kg TOCP for 90 days. In a third experiment, animals were given single oral doses of 2500 mg/kg triallate, 750 mg/kg TOCP, or empty gelatin capsules and killed after 24 hr. Delayed neurotoxicity was observed only in TOCP-treated animals. Animals given daily doses of 300 mg/kg triallate became moribund after 30 days; however, histological examination revealed no lesions characteristic of organophosphorus-induced delayed neurotoxicity. Neurotoxic esterase was not significantly altered in triallate-treated animals while it was 95% inhibited in TOCP-treated animals. Plasma butyrylcholinesterase increased significantly 24 hr after treatment with triallate in a dose-dependent manner. In summary, triallate, a thiocarbamate, did not produce neurotoxicity which has been previously reported for some dithiocarbamates.

Purification and characterization of cytochrome P-450 isozymes from phenobarbital-induced adult hen liver.

1. Two cytochrome P-450 isozymes (P-450 PB-A, PB-B) and cytochrome b5 were purified from livers of phenobarbital-treated adult hens. 2. Both the enzymes exhibited the same apparent molecular weight (54,000). 3. They could be distinguished on the basis of immunochemical properties, spectral properties, peptide pattern after partial proteolysis, tryptic peptide pattern, and N-terminal sequence. 4. The antibodies raised against P-450 PB-A and PB-B did not cross-react with microsomal P-450s of rat, mice, cat, or catfish species by immunoblotting.

Mechanisms of organophosphorus ester-induced delayed neurotoxicity: type I and type II.

Some organophosphorus compounds produce neurologic dysfunctions, known as OPIDN, after a delay period that is accompanied by neuropathic damage in the central and peripheral nervous systems. This group of chemicals may be divided into two classes, Type I and II, based on chemical structure, species selectivity, age sensitivity, the length of latent period, clinical signs, morphology and distribution of neuropathologic lesions, protection with phenylmethyl sulfonyl fluoride, inhibition of neurotoxic esterase, and effect on catecholamine secretion from bovine adrenome-dullary chromaffin cells. The importance of this effect is underlined by the fact that incidents involving more than 40,000 cases of OPIDN in humans have been documented from 1899 to 1989. Most of these compounds are direct or indirect inhibitors of AChE, and produce acute cholinergic effects. Neurologic deficits are characterized by three phases: progressive, stationary, and improvement. Prognosis of OPIDN depends on the extent of damage of the nervous system. Improvement or even recovery of functions may follow mild cases, whereas severe toxicity results in long-lasting neurologic dysfunctions reflecting spinal cord damage. Recent studies have shown that delayed neurotoxic organophosphorus compounds interact with Ca2+/calmodulin kinase II (CaM kinase II), an enzyme responsible for the endogenous phosphorylation of cytoskeletal proteins, i.e. microtubules, neurofilaments, and MAP-2. This leads to an increased activity of CaM kinase II and enhanced phosphorylation of cytoskeletal elements, and eventually in the disassembly of cytoskeletal proteins. The dissociation of cytoskeletal proteins causes increased fast axonal transport in the treated animals resulting in the accumulation of altered cytoskeletal elements in the distal portions of the axon. Abnormal tubulin and neurofilaments are transformed into filamentous polymers and undergo condensation and dissolution. Concomitantly, proliferated endoplasmic reticulum and accumulated mitochondria degenerate and release Ca2+ ions. This leads to Ca2(+)-activated proteolysis of the cytoskeleton and interruption of ionic balance across the axonal membrane resulting in the uptake of water and axonal swelling, which subsequently degenerates. A similar mechanism may cause secondary myelin degeneration.

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.

Acceleration of anterograde axonal transport in cat sciatic nerve by diisopropyl phosphorofluoridate.

The effect of a neurotoxic dose of 5.0 mg/kg, s.c. diisopropyl phosphorofluoridate (DFP) on anterograde transport of 35S-methionine labeled proteins in cat peripheral nerve was studied. Seven days after dosing, after 24 h of flow there was 48% less radioactivity in the distal portion of the nerve. A lesser effect was also found at 4 and 14 days after dosing. After 10 or 14 h of flow, the height of the crest was unchanged, but the distance of the crest from the ganglia was greater in the DFP-treated animals. These experiments indicate that DFP treatment accelerates fast anterograde transport.