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 [gamma-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.

Brainstem Axolemmal Protein Phosphorylation in Vitro in Hens Dosed With Di-1-Butyl-2,2-Dichlorovinyl Phosphate

“…Previously reported work has also shown disrupted endogenous protein phosphorylation following the development of clinical signs of OPIDN (Patton et al, 1985), but more recent work has shown changes as early as d 1 following dosing with TOCP (Lapadula et al, 1991). Moreover, although the present study consistently found a decrease of about 30% in the P 50 band from brainstem axolemmal membranes, other work (Patton et al, 1985;Lapadula et al, 1991) has found large increases in several cytosolic and membrane proteins from brain and spinal cord. Possible reasons for these different findings include the fact that DBDCV is much more potent than TOCP in producing OPIDN and could possibly exert a more selective effect on protein phosphorylation.…”

“…In particular, this research highlights the notion that doses of different OP compounds or other neuropathic agents that yield comparable clinical deficits are not necessarily equivalent with respect to the spatiotemporal evolution of the damage produced or the specific molecular targets involved. As suggested previously (Richardson, 1984;Patton et al, 1985;Saitoh et al, 1991;Abou-Donia, 1993), it appears likely that continued studies of the impact of neurotoxicants on endogenous protein phosphorylation will provide valuable indicators of the degree and type of cellular and molecular disruption produced in a given nervous system locus, and in some cases may also lead to an understanding of molecular pathogenesis.…”

“…This apparent disparity could be due to a number of reasons. The dose of TOCP may not have been high enough; for example, Patton et al (1985) did observe changes in endogenous phosphorylation after 750 mg/kg TOCP. However, as indicated earlier, the changes observed by these authors were not in axolemmal fractions, and they used different conditions for studying protein phosphorylation.…”

“…1969a; Lush et al, 1998), endogenous protein phosphorylation (Huggins & Richardson, 1982;Patton et al, 1985), microtubule assembly (Seifert & Casida, 1982), retrograde axonal transport (Moretto et al, 1987), and calcium-activated neutral protease (El-Fawal et al, 1990). All of these targets are affected to varying degrees in neural tissues by different neuropathic OP compounds in several animal species.…”

Sarin (GB, O-isopropyl methylphosphonofluoridate) neurotoxicity: critical review

“…An early event in OPIDN is an increased Ca2+ concentration in neuronal mitochondria of the hens’ spinal cord (LoPachin et al 1988). This increase is followed by enhanced auto-phosphorylation (Patton et al 1983, 1985, 1986), activity (Lapadula et al 1991, 1992; Abou-Donia et al 1993) and mRNA expression (Gupta et al 1998) of CaM Kinase II. Activated CaM kinase II causes hyperphosphorylation of the cytoskeletal proteins: MAP-2; (Patton et al 1983, 1985, 1986), tau (Gupta & Abou-Donia 1999), α- and β-tubulin (Gupta & Abou-Donia 1994; Suwita et al 1986a, 1986b; Suwita & Abou-Donia 1990), neurofilament triplet proteins (Gupta & Abou-Donia 1995a; Gupta et al 1999) and myelin basic protein (Abou-Donia 1995).…”