Screening for developmental neurotoxicity using PC12 cells: comparisons of organophosphates with a carbamate, an organochlorine, and divalent nickel

Abstract

Background: In light of the large number of chemicals that are potential developmental neurotoxicants, there is a need to develop rapid screening techniques.

Objectives: We exposed undifferentiated and differentiating neuronotypic PC12 cells to different organophosphates (chlorpyrifos, diazinon, parathion), a carbamate (physostigmine), an organochlorine (dieldrin), and a metal (divalent nickel; Ni2+) and examined indices of cell replication and differentiation for both short- and long-term exposures.

Results: In undifferentiated cells, all the agents inhibited DNA synthesis, with the greatest effect for diazinon, but physostigmine eventually produced the largest deficits in the total number of cells after prolonged exposure. The onset of differentiation intensified the adverse effects on DNA synthesis and changed the rank order in keeping with a shift away from noncholinergic mechanisms and toward cholinergic mechanisms. Differentiation also worsened the effects of each agent on cell number after prolonged exposure, whereas cell growth was not suppressed, nor were there any effects on viability as assessed with trypan blue. Nevertheless, differentiating cells displayed signs of oxidative stress from all of the test compounds except Ni2+, as evidenced by measurements of lipid peroxidation. Finally, all of the toxicants shifted the transmitter fate of the cells away from the cholinergic phenotype and toward the catecholaminergic phenotype.

Conclusions: These studies point out the feasibility of developing cell-based screening methods that enable the detection of multiple end points that may relate to mechanisms associated with developmental neurotoxicity, revealing some common targets for disparate agents.

Figure 1

DNA synthesis shown as disintegrations per minute (dpm; mean ± SE) of [³H]thymidine in (A) undifferentiated cells exposed to 5 μM of agent, (B) undifferentiated cells exposed to 30 μM, and (C) differentiating cells exposed to 30 μM and cotreated with NGF (note that scale in C differs from that in A and B). ANOVA across all treatments and time points (number of determinations for each condition): (A), treatment, p < 0.0001; treatment × time, p < 0.0001 (n = 6–18); (B), treatment, p < 0.0001; treatment × time, p < 0.0001 (n = 12–30); (C), treatment, p < 0.0001; treatment × time, p < 0.0001 (n = 12–30). *Significantly different from the corresponding control value (p < 0.05).

Figure 2

Indices of cell number and size (mean ± SE) in undifferentiated cells exposed to 30 μM of agent for 6 days. (A) DNA content. (B) Total protein/DNA ratio. (C) Membrane/total protein ratio. ANOVA across all treatments (number of determinations for each condition): (A) p < 0.0001 (n = 5–10); (B) p < 0.0001 (n = 5–10); (C) p < 0.0004 (n = 5–10). *Significantly different from the corresponding control value (p < 0.05).

Figure 3

Indices of cell number and size (mean ± SE) in differentiating cells exposed to 30 μM of agent and cotreated with NGF. (A) DNA content. (B) Total protein/DNA ratio. (C) Membrane/total protein ratio (note the interrupted ordinate scale). ANOVA across all treatments and both time points (number of determinations for each condition): (A) treatment, p < 0.0001; treatment × time, p < 0.0001 (n = 10–22); (B) treatment, p < 0.0001; treatment × time, p < 0.0001 (n = 10–22); (C) treatment, p < 0.004; treatment × time, p < 0.0001 (n = 10–22). *Significantly different from the corresponding control value (p < 0.05).

Figure 4

Indices of viability, oxidative damage, and transmitter phenotype (mean ± SE) in differentiating cells exposed to 30 μM of agent and cotreated with NGF for 6 days. (A) Trypan blue staining. (B) TBARS (note the interrupted ordinate scale). (C) TH/ChAT activity ratio. ANOVA across all treatments (number of determinations for each condition) (A) not significant (n = 10–22); (B) p < 0.0001 (n = 5–10); (C) p < 0.0001 (n = 10–25). *Significantly different from the corresponding control value (p < 0.05).