Comparative developmental neurotoxicity of organophosphate insecticides: effects on brain development are separable from systemic toxicity

Abstract

A comparative approach to the differences between systemic toxicity and developmental neurotoxicity of organophosphates is critical to determine the degree to which multiple mechanisms of toxicity carry across different members of this class of insecticides. We contrasted neuritic outgrowth and cholinergic synaptic development in neonatal rats given different organophosphates (chlorpyrifos, diazinon, parathion) at doses spanning the threshold for impaired growth and viability. Animals were treated daily on postnatal days 1-4 by subcutaneous injection so as to bypass differences in first-pass activation to the oxon or catabolism to inactive products. Evaluations occurred on day 5. Parathion (maximum tolerated dose, 0.1 mg/kg) was far more systemically toxic than was chlorpyrifos or diazinon (maximum tolerated dose, 1-5 mg/kg). Below the maximum tolerated dose, diazinon impaired neuritic outgrowth in the forebrain and brainstem, evidenced by a deficit in the ratio of membrane protein to total protein. Diazinon also decreased choline acetyltransferase activity, a cholinergic neuronal marker, whereas it did not affect hemicholinium-3 binding to the presynaptic choline transporter, an index of cholinergic neuronal activity. There was no m(subscript)2(/subscript)-muscarinic acetylcholine receptor down-regulation, as would have occurred with chronic cholinergic hyperstimulation. The same pattern was found previously for chlorpyrifos. In contrast, parathion did not elicit any of these changes at its maximum tolerated dose. These results indicate a complete dichotomy between the systemic toxicity of organophosphates and their propensity to elicit developmental neurotoxicity. For parathion, the threshold for lethality lies below that necessary for adverse effects on brain development, whereas the opposite is true for chlorpyrifos and diazinon.

Figure 1

Effects of different organophosphates on mortality during daily treatment on PND1–4 and for up to 24 hr after the last dose. Data represent cumulative mortality obtained from a minimum of 60 animals in each treatment group at each age. Data for 5 mg/kg chlorpyrifos were compiled from previous results (Campbell et al. 1997; Whitney et al. 1995). *p < 0.05 compared with control, Fisher’s exact test.

Figure 2

Effects of different doses of diazinon and parathion on the membrane protein:total protein ratio in brainstem (bs) and forebrain (fb), assessed on PND5 and presented as the percent change from the corresponding control values (Table 1). ANOVA across all treatments, both regions and both sexes: main treatment effect, p < 0.0001. *Significantly different (p < 0.05) from corresponding control values; statistical significance for individual regions or sexes was not determined because of the absence of treatment × region and treatment × sex interactions.

Figure 3

Effects of different doses of diazinon and parathion on markers of cholinergic synaptic development in brainstem (bs) and forebrain (fb), assessed on PND5. (A) ChAT activity (ANOVA: treatment, p < 0.0001). (B) m₂AChR receptor binding (ANOVA: treatment, p < 0.03; treatment × sex, p < 0.0002; male, p < 0.0001. (C) HC3 binding to the high-affinity presynaptic choline transporter (ANOVA: treatment × sex × region, p < 0.05). Data are presented as the percent change from the corresponding control values (Table 1). *Significantly different (p < 0.05) from corresponding control values; statistical significance for individual regions or sexes was determined only where there were corresponding treatment × region or treatment × sex interactions.