Elsevier

Neuroscience

Volume 146, Issue 1, 25 April 2007, Pages 330-339
Neuroscience

Neuropharmacology
Microtubule-associated targets in chlorpyrifos oxon hippocampal neurotoxicity

https://doi.org/10.1016/j.neuroscience.2007.01.023Get rights and content

Abstract

Prolonged exposure to organophosphate (OP) pesticides may produce cognitive deficits reflective of hippocampal injury in both humans and rodents. Recent work has indicated that microtubule trafficking is also adversely affected by exposure to the OP pesticide chlorpyrifos, suggesting a novel mode of OP-induced neurotoxicity. The present studies examined effects of prolonged exposure to chlorpyrifos oxon (CPO) on acetylcholinesterase (AChE) activity, immunoreactivity (IR) of microtubule-associated proteins, neuronal injury, and tubulin polymerization using in vitro organotypic slice cultures of rat hippocampus and bovine tubulin. Cultures were exposed to CPO (0.1–10 μM) in cell culture medium for 1–7 days, a regimen producing progressive reductions in AChE activity of 15–60%. Cytotoxicity (somatic uptake of the non-vital marker propidium iodide), as well as IR of α-tubulin and microtubule-associated protein-2 (a/b) [MAP-2], was assessed 1, 3, and 7 days after the start of CPO exposure. As early as 24 h after the start of exposure, CPO-induced deficits in MAP-2 IR were evident and progressive in each region of slice cultures at concentrations as low as 0.1 μM. CPO exposure did not alter α-tubulin IR at any time point. Concentration-dependent injury in the cornu ammonis (CA)1 pyramidal cell layer and to a lesser extent, CA3 and dentate cells, was evident 3 days after the start of CPO exposure (≥0.1 μM) and was greatest after 7 days. Tubulin polymerization assays indicated that CPO (≥0.1 μM) markedly inhibited the polymerization of purified tubulin and MAP-rich tubulin, though effects on MAP-rich tubulin were more pronounced. These data suggest that exposure to CPO produces a progressive decrease in neuronal viability that may be associated with impaired microtubule synthesis and/or function.

Section snippets

Organotypic hippocampal slice preparations

Complete brains from 8-day-old male and female Sprague–Dawley rat pups were aseptically extracted and transferred to dissection medium (4 °C), consisting of Minimum Essential Medium (MEM) plus 25 mM Hepes, 2 mM l-glutamine, and 50 μM streptomycin/penicillin. Bilateral hippocampi were dissected out and placed into chilled culture medium. Culture medium consisted of dissection medium with the addition of sterile H2O, 36 mM glucose, 25% Hanks’ balanced salt solution (HBSS), and 25%

AChE activity

Following 1 day of exposure to the two highest concentrations of CPO (1.0 and 10.0 μM), AChE activity in the slice cultures was reduced by approximately 50% [F(3,206)=107.86, P<0.001], though no marked differences in AChE activity between these two groups were noted (post hoc=P<0.05 vs. control and 0.1 μM). Exposure to the 0.1 μM concentration of CPO did not reduce AChE activity at this time point. In contrast, 3 days of exposure to CPO, at each concentration, markedly reduced AChE activity [F

Discussion

The present studies were designed to examine the effects of prolonged exposure to CPO on AChE activity, as well as, the bioavailability of microtubule-related proteins α-tubulin and MAP-2a/b in a neonatal rat slice culture preparation. Further, these studies examined effects of CPO exposure on these proteins in parallel with the time course of cytotoxicity. Lastly, effects of CPO exposure on polymerization of purified and MAP-rich bovine tubulin were assessed to examine whether CPO affected

Conclusion

In conclusion, the present findings are consistent with previous work and extend our understanding of the means by which CPF and CPO produce neuronal injury that is independent of AChE inhibition and may involve degradation of microtubules, possibly mediated by interactions with MAP-2 proteins. Thus, it is possible that prolonged CPF exposure in vivo may significantly impact not only early CNS development but neurogenesis in the adult brain.

Acknowledgments

This work was supported by NIEHS (ES012241).

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