Expression changes of neurofilament subunits in the central nervous system of hens treated with tri-ortho-cresyl phosphate (TOCP)

Abstract

Tri-ortho-cresyl phosphate (TOCP) could induce degeneration of long, large diameter axons within the central and peripheral nervous system of susceptible species including human being and hens, which is referred to as organophosphorus-ester induced delayed neuropathy (OPIDN). The mechanisms involved are not understood. Neuropathologic observations suggested that neurofilament subunits (NFs) could be a main target of TOCP in the peripheral nervous system. Our previous study also showed that NFs in protein levels significantly decreased in sciatic nerves of hens treated with TOCP. In this study, to determine whether the decrement of NFs proteins in sciatic nerves was due to reductions in NF gene expression or protein degradation, hens were treated with a single dose of 750 mg/kg body weight TOCP by gavage, and sacrificed on 21 day post-exposure. Cerebral cortexes and spinal cords were sampled. Transcriptional changes of NFs including high molecular weight neurofilament (NF-H), middle molecular weight neurofilament (NF-M), low molecular weight neurofilament (NF-L), and glyceraldehydes-3-phoaphate dehydrogenase (GAPDH) as inner inference in cerebral cortexes and spinal cords were analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results showed that all of three NFs mRNA in cerebral cortexes down-regulated significantly. However, in spinal cords, there was only NF-M decreased, both of NF-H and NF-L kept unaffected. The protein levels of NFs in pellet and supernatant fractions of cerebral cortexes and spinal cords were also determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We noticed that all NFs protein declined in pellet of cerebral cortexes, but NF-M reduction was not significant compared with that of control hens. NF-H and NF-M proteins in supernatant of cerebral cortexes exhibited significant increase, while NF-L level showed remarkable decline. In spinal cords, apart from NF-L in pellet were significantly increased, both of NF-H and NF-M in pellet and supernatant, as well as NF-L in supernatant fractions were manifested dramatic reduction compared with the pattern of control. The quantitative analyses revealed that the change magnitude in protein levels was much greater than that in mRNA levels in hens' central nervous system after TOCP administration. These findings suggest that the NFs disturbance in protein levels is closely associated with the decreases in sciatic nerves observed in our previous work after TOCP exposure, rather than that in mRNA levels, and the NFs alterations in protein levels may be one of the responsible factors for the OPIDN.