Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish

Abstract

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10-30 nM DDT, 0.3-3 μM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging.

Keywords: Aging; Anxiety-related behavior; DDT; Neurobehavioral toxicology; Zebrafish.

Fig. 1.

Set 1 Results. Two weeks of DDT exposure at 30nM suppressed the average locomotor activity in the 5 sec prior to the tap stimulus (mean ± sem) at 1-week post-exposure. Asterisk (*) indicates significant difference from controls at the p < 0.05 level.

Fig. 2.

Set 2 Results: 1-week post-exposure. Five weeks of DDT exposure led to multiple effects at 1-week post-exposure (mean +/− SEM). Treatment with 30nM DDT led to reduced locomotion in the latter half of the novel tank diving test at multiple individual time points (A), a reduced average distance from the bottom in the novel tank, collapsed across time points (B), as well as reduced activity during the first presentation of the fast stimulus (C) and increased distance from the fast predator stimulus (D). Asterisk (*) indicates significant difference from controls at the p < 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10.

Fig. 3.

Set 2 Results: 14 months of age. Five weeks of DDT exposure led to multiple effects at 14 months of age (mean +/− SEM). Prior treatment with 30nM DDT significantly increased startle magnitudes to the initial taps in the tap test (A). Prior exposure to 10nM DDT led to a reduced total locomotor activity in the shoaling assay (B). Asterisk (*) indicates significant difference from controls at the p < 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10.

Fig. 4.

Neurochemistry results: DDT. Neurochemical effects of DDT exposure (mean +/− SEM): (A) ChAT activity, (B) HC3 binding. There were no significant treatment effects on either parameter.

Fig. 5.

Set 3 Results: 1 week post-exposure. Two weeks of CPF exposure led to multiple effects at 1-week post-exposure (mean +/− SEM). Treatment with 3.0uM CPF led to increased average distance from the bottom in the novel tank, collapsed across time points (A) and the magnitude of the approach response in the shoaling assay (B). Asterisk (*) indicates a significant difference from controls at the p < 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10.

Fig. 6.

Set 4 Results. Five weeks of moderate CPF exposure led to multiple effects at 14 months of age (mean +/− SEM). Treatment with 3.0uM CPF altered distance from the screen at 1-week post-exposure (A). By contrast at 14 months, 0.3uM CPF led to reduced distance to the screen following removal of the fast predator stimulus (B), increased locomotor activity overall in the predator avoidance assay (C) and increased approach of the screen after the slow stimulus was removed (D). Asterisk (*) indicates significant difference from controls at the p < 0.05 level.

Fig. 7.

Neurochemistry results: CPF. Neurochemical effects of CPF exposure (mean +/− SEM): (A) ChAT activity, (B) HC3 binding. ChAT was unaffected by CPF, but HC3 binding was significantly reduced.

Fig 8.

Age-related effects on behavior. Pooled analysis of controls indicated that older fish showed higher average levels of locomotion relative to younger fish in the novel tank dive test, collapsed across time points (A). No age effects were evident on tap test performance (B), or during the shoaling assay (C). In the predator avoidance assay, older fish remained closer to the video screen (left axis), both when the predator cues were present and absent, as well as showed a reduced avoidance score, measured as a change in position after the predator cue was removed (right axis) (D). Asterisk (*) indicates significant difference from controls at the p < 0.05 level.