Mohamed B. Abou-Donia - US grants

DESCRIPTION: (Adapted from the Investigator's Abstract) The objective of this proposal is to investigate the molecular mechanisms of organophosphorus compound induced delayed neurotoxicity (OPIDN). OPIDN is a central-peripheral distal axonopathy. Early ultrastructural alterations in OPIDN are characterized by the presence in the distal axon of aggregated cytoskeletal proteins; microtubules, and neurofilaments. The investigators have demonstrated that central to the pathogenesis of OPIDN is an anomalous increase in Ca2+/calmodulin-dependent kinase- mediated phosphorylation of the cytoskeletal proteins, i.e. neurofilament triplet proteins, and tublin, microtubule associated protein-2 (MAP-2), and tau protein. Although in OPIDN there is an enhanced activity and autophosphorylation of Ca2+/calmodulin dependent kinase II (CAM kinase II), this enzyme, however, does not seem to account for all of the increase in Ca2+/calmodulin-dependent phosphorylation of cytoskeletal proteins. In addition, other kinases are likely to be involved. Consistent with enhanced phosphorylation of cytoskeletal proteins are the PIs recent findings of the increase in CaM kinase II mRNA expression and slow axonal transport of radiolabeled neurofilament proteins in OPIDN. In this proposal, the investigators propose to investigate the hypothesis that sustained and prolonged hyperphosphorylation of cytoskeletal proteins results in an exaggeration of normal phosphorylation and induced conformational changes leading to their aggregation and impairment of vital axonal processes such as axonal transport with subsequent degeneration of the axon. There are four elements in this approach: 1) identification and characterization of the kinases that may be involved in the conversion of normal cytoskeletal proteins into the "hyperphosphorylated" state and assessment of the possibility that aberrantly activated kinases act in concert to generate abnormally phosphorylated cytoskeletal proteins; 2) delineation of the time-course and extent of phosphorylation of amino acid residues that are hyperphosphorylated in cytoskeletal proteins; 3) determination of functional, structural, and biochemical consequences of the transformation of cytoskeletal proteins; and 4) investigation of the time-course and mechanisms of transcriptional alterations in cytoskeletal proteins, associated kinases, and immediate early genes. Taken together, they plan to identify the differences between normal and pathological cytoskeletal proteins and assess how hyperphosphorylation of these proteins disrupts their assembly and compromises their stability, leading to functional alterations and axonal degeneration.

Many chemicals produce neurotoxicity in humans and some animal species. Toxicological studies and epidemiological evidence implicated that simultaneous exposure to the organophosphorus insecticide leptophos and the industrial solvents, n-hexane and toluene, caused an outbreak of neuropathy among workers handling these chemicals. Leptophos belongs to a group of chemicals capable of causing delayed neurotoxicity (OPIDN). The adult hen is the animal of choice to study OPIDN since rodents are not as sensitive. n-Hexane and some of its related chemicals produce neurotoxicity in humans and in animals. Toluene has been implicated in causing neurologic deficits in humans. Because humans are often exposed concurrently to chemicals from these groups, we have been studying the neurotoxicity produced by these chemicals alone and in combination. During the first phase of these studies we have tested the hen as a laboratory animal to investigate the neurotoxicity produced by simultaneous administration of the organophosphorus insecticide EPN (O-ethyl 0-4-nitrophenyl phenylphosphonothioate) and n-hexane, 2-hexanone (MnBK), 2,5-hexanediol (2,5-HDOH), and 2,5-hexanedione (2,5-HD). The results showed that simultaneous exposure to EPN and MnBK potentiated neurotoxicity produced by each chemical alone. Also, the non-neurotoxic methyl iso-butyl ketone (MiBK) synergized the neurotoxicity of the weak neurotoxicant n-hexane. The mechanism of this joint neurotoxic action seems to be related, at least in part, to the induction of liver microsomal cytochrome P-450. We propose to investigate the molecular mechanism(s) for aliphatic hexacarbon-induced neurotoxicity and liver microsomal enzyme induction by these neurotoxicants. This will include the isolation of the cross-linked neurofilament proteins from hens exposed to vapors of n-hexane/MiBK and the use of these proteins as markers for aliphatic hexacarbon neurotoxicity and neurotoxic esterase for EPN-induced neurotoxicity. The cytochrome P-450 form(s) that is inducible by hen exposure to n-hexane related chemicals and EPN alone and together will be determined in hen liver microsomes. The in vitro metabolism of [14C]labeled n-hexane, 2,5-HDOH, 2,5-HD, and EPN by chicken hepatic microsomes will be determined. Absorption and pharmacokinetics of a single dermal dose of [14C]EPN following repeated doses of n-hexane and its metabolites will be studied. The neurotoxicity of toluene vapor will be determined following inhalation by chickens. The joint neurotoxic action of toluene, n-hexane, and/or EPN will be investigated.

Some organophosphorus (OP) compounds produce delayed neurotoxicity (OPIDN). The adult chicken is the test animal for OPIDN. This effect is manifested as ataxia followed by paralysis resulting in Wallerian-type degeneration of the central and peripheral nervous systems. Early histopathologic changes are aggregation of neurotubules and neurofilaments followed by their condensation. Preliminary studies have suggested that Ca2+ is the initiating factor, subsequent to protein phosphorylation, in the development of OPIDN. It is planned to study the proposed mechanism set forth for OPIDN. It is proposed that neurotoxic OPs phosphorylate a neurotoxicity target protein whose normal function is unknown but could be related to calcium homeostasis. The subsequent increase in Ca2+ brings about depolymerization of microtubules to tubulins and an increased Ca2+-calmodulin-dependent phosphorylation of tubulin. As a result, tubulin is aggregated. Accumulation of such structures leads to the disruption of axoplasmic transport and the accumulation of mitochondria at the distal parts of the axons. Broken down mitochondria release Ca2+ into the axoplasm. This disrupts axonal membrane mechanisms for intracellular/extracellular ionic gradient, which leads to focal internodal swelling and degeneration that spreads stomatofugally to involve the entire distal axon. We propose to study the effect of the neurotoxic OP, DFP, in comparison with the non-neurotoxic parathion on the normal phosphorylating mechanisms of neuronal proteins and on Ca2+ concentration of the axoplasm of neurons. Similar experiments will be conducted on chicks and rats (insensitive to OPIDN). The effect of DFP or intracellular Ca++ concentration will be studied using biochemical and electrophysiological techniques. The effect of delayed neurotoxic versus non-delayed neurotoxic OPs administered in vivo or incubated with samples in vitro, on Ca++ uptake into synaptosomal, microsomal and mitochondrial fractions will be investigated using 45Ca++. Detection of free cytosolic Ca++ will be studied using Arsenazo III to monitor alterations in free cytosolic Ca++ after OP treatment. The kinetics of polymerization and depolymerization of microtubules in the presence of Ca++, OPs, calmodulin, and tubulin associated calmodulin kinase (TACK) will be studied. The isolation of TACK from treated and untreated animals will be used to investigate if this kinase is directly affected by OP treatment. Electrophysiological recordings of alterations in internal Ca++ concentration in Aplysia before and after OP treatment will be investigated.

The goal of this proposal is to examine the mechanism(s) through which covalent binding of neurotoxicants (acrylamide, carbon disulfide (CS2), 2,5-hexanedione (2,5-HD)), to cytoskeletal proteins produces a dying back type of neuropathy. The present study is motivated by the pathological findings that many compounds which covalently bind with specific amino acid residues produce a dying back type of neuropathy. Common neuropathological features of many of these types of neuropathies include axonal swellings which may contain a high number of neurofilaments. Understanding the underlying mechanism which produces this type of neuropathy is primarily important for the elucidation of chemically induced injury to nerve cells. The hypothesis of this proposal is that acrylamide, carbon disulfide, and aliphatic hexacarbons directly interfere with neurofilament function. Several possible alterations of neurofilament biochemistry will be investigated. The mechanisms through which these alterations occur involve a direct binding of the toxicant to neurofilament proteins, an inhibition of protein phosphorylation, an inhibition of proteolytic proteins. The specific aims of this proposal are (1) to examine the in vitro and in vivo covalent binding of [14C]labeled neurotoxicant to cytoskeletal and identification of the amino acid residue which is bound, (2) to study in vitro protein phosphorylation after in vitro incubation with neurotoxicants or in vivo exposure to neurotoxicants, (3) to study in vitro and in vivo proteolytic breakdown of neurofilaments, (4) to examine the reconstitution of neurofilaments after in vitro exposure of neurofilaments to chemicals, (5) to study the interaction of neurofilaments with microtubules and microtubule associated proteins in vitro after in vivo exposure of cytoskeletal proteins, (6) Quantitate change in specific epitopes of neurofilament proteins (i.e. phosphorylated versus de-phosphorylated epitopes) after exposure to these chemicals, and (7) to determine if a protein kinase which is associated with neurofilaments is specifically altered by vivo treatment or in vitro incubation with these neurotoxicants.