Developmental neurotoxicity of pyrethroid insecticides in zebrafish embryos

Abstract

Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

FIG. 1.

Representative structures of pyrethroid pesticides tested for developmental toxicity.

FIG. 2.

Experimental timeline for dosing and pharmacological interventions.

FIG. 3.

Developmental exposure of zebrafish to pyrethroid pesticides results in a dose-related increase in mortality. Zebrafish were exposed to pyrethroid pesticides at various doses for 6 dpf and observed for mortality every 24 h. Data are presented as mean ± SEM. Percent mortality values greater than 20% (gray dotted line) were statistically significant from controls. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. BF, bifenthrin; CH, ∧-cyhalothrin; CM, cypermethrin; DM, deltamethrin; PM, permethrin; RM, resmethrin.

FIG. 4.

Developmental exposure of zebrafish to high doses of pyrethroid pesticides causes pericardial edema. Zebrafish were exposed to pyrethroid pesticides at various doses for 6 dpf and observed for edema every 24 h. Data are presented as mean ± SEM. Percent edema for control groups ranged from 0 to 11%. Percent lesion values greater than 20% (gray dotted line) were statistically significant from controls. Picture below shows representative pericardial edema. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. BF, bifenthrin; CH, ∧-cyhalothrin; CM, cypermethrin; DM, deltamethrin; PM, permethrin; RM, resmethrin.

FIG. 5.

Developmental exposure of zebrafish to high doses of pyrethroid pesticides alters crainiofacial structure. Pictures show representative craniofacial appearance of vehicle control and permethrin- or deltamethrin-treated zebrafish under ×40 magnification, with doses in units of micrograms per liter. Zebrafish were exposed beginning 3 hpf. Larvae were fixed at 6 dpf and stained with alcian blue dye to allow visualization of cartilage. Images shown are from the same experimental run and show the typical appearances of fish exposed to permethrin or deltamethrin. Alcian blue staining was performed on three experiments for both compounds and significantly different groups are indicated with boxes. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. DM, deltamethrin; PM, permethrin.

FIG. 6.

Developmental exposure of zebrafish to various pyrethroids causes curvature of the body axis. Zebrafish were exposed to pyrethroid pesticides at various doses for 6 dpf and observed for curvature. Data are presented as mean ± SEM. Percent lesion values greater than 20% (gray dotted line) were found to be statistically greater than control. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. BF, bifenthrin; CH, ∧-cyhalothrin; CM, cypermethrin; DM, deltamethrin; PM, permethrin; RM, resmethrin.

FIG. 7.

Developmental exposure to pyrethroid pesticides causes a dose-dependent increase in spasms. Zebrafish were exposed to pyrethroid pesticides at various doses beginning at 3 hpf and observed for spasms at 96 hpf. Data are presented as mean ± SEM. Percent lesion values greater than 20% (gray dotted line) were statistically significant from control. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. BF, bifenthrin; CH, ∧-cyhalothrin; CM, cypermethrin; DM, deltamethrin; PM, permethrin; RM, resmethrin.

FIG. 8.

Sodium channel blockade with MS-222 prevents spasms and curvature induced by developmental deltamethrin exposure. Zebrafish were exposed to deltamethrin (25 μg/l) from 3 hpf until 72 hpf. Embryos were dechorionated at 48 hpf and dosed with MS-222. Data shown are from observations at 72 hpf or 24 h after MS-222 dosing. Significant differences (p < 0.001) from control are indicated with asterisks. Control animals exposed to MS-222 without deltamethrin demonstrated no lesions and, at the highest dose tested, were still able to respond to a touch stimulus at 72 hpf. (Right) Representative pictures taken at 72 hpf, at which point, fish had been coexposed to deltamethrin (25 μg/l) and MS-222 (or control) for 24 h. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times. DM, deltamethrin.

FIG. 9.

The GABA_A receptor antagonist diazepam ameliorates spasms but not body axis curvature induced by developmental deltamethrin exposure. Animals were exposed to 25 μg/l deltamethrin beginning at 3 hpf. Animals were dosed with diazepam at 72 hpf and reobserved 2 h after dosing. Significant differences (p < 0.001) are indicated by asterisks. Diazepam had no significant effect on the incidence of body axis curvature at any of the doses tested. Each experiment comprised 20–25 fish per treatment group, and each experiment was repeated at least three times.