A critical review of neonicotinoid insecticides for developmental neurotoxicity

<div><p></p><p>A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood–brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified <i>in vitro</i>, <i>in vivo</i>, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system.</p></div

Dietary Administration of Paraquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the \u3cem\u3eSubstantia nigra\u3c/em\u3e of C57BL/6J Mice

Several investigations have reported that mice administered paraquat dichloride (PQ·Cl2) by intraperitoneal injection exhibit a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In this study, male and female C57BL/6J mice were administered PQ·Cl2 in the diet at concentrations of 0 (control), 10, and 50 ppm for a duration of 13 weeks. A separate group of mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) during week 12 as positive controls to produce a loss of dopaminergic neurons in the SNpc. The comparative effects of PQ and MPTP on the SNpc and/or striatum were assessed using neurochemical, neuropathological, and stereological endpoints. Morphological and stereological assessments were performed by investigators ‘blinded’ to the origin of the tissue. Neither dose of PQ·Cl2 (10 or 50 ppm in the diet) caused a loss of striatal dopamine or dopamine metabolite concentrations in the brains of mice. Pathological assessments of the SNpc and striatum showed no evidence of neuronal degeneration or astrocytic/microglial activation. Furthermore, the number of tyrosine hydroxylase-positive (TH+) neurons in the SNpc was not reduced in PQ-treated mice. In contrast, MPTP caused a decrease in striatal dopamine concentration, a reduction in TH+ neurons in the SNpc, and significant pathological changes including astrocytic and microglial activation in the striatum and SNpc. The MPTP-induced effects were greater in males than in females. It is concluded that 13 weeks of continuous dietary exposure of C57BL/6J mice to 50 ppm PQ·Cl2 (equivalent to 10.2 and 15.6 mg PQ ion/kg body weight/day for males and females, respectively) does not result in the loss of, or damage to, dopaminergic neurons in the SNpc

Stereological assessment of the mean number of TH⁺ neurons in the SNpc following PQ or MPTP treatment in C57BL/6J and C57BL/6NHsd male mice.

<p>Five different groups (G1-G5) of animals, varying in age and site of experiment were injected with saline, paraquat or MPTP and the extent of TH⁺ neuron loss was assessed by design-based or model-based stereology. ** significantly different from control mice (p ≤ 0.01). Syngenta-sourced PQ was used to treat mice G1 to G3 mice, whereas Sigma Chemical PQ was used to treat mice in groups G4 and G5.</p

Correlation between the number of activated microglia in the SNpc and the number of TH⁺ neurons.

<p>The correlation between the number of activated microglia in the SNpc and the number of TH⁺ neurons was assessed in the same animal in two studies on C57BL/6J mice (A, B) and one study using C57BL/6NHsd (C) performed at two different sites (WIL, A) and SJCRH (B, C). A significant negative inverse correlation between TH⁺, DA neuron number and activated microglia was observed in MPTP-treated mice, while no correlation was seen in PQ- or saline treated mice.</p

Number, design and location of studies examining effects of paraquat and MPTP on C57BL/6J and C57BL/6NHsd male mice (aged 9 or 16 weeks)¹.

<p>Number, design and location of studies examining effects of paraquat and MPTP on C57BL/6J and C57BL/6NHsd male mice (aged 9 or 16 weeks)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164094#t001fn001" target="_blank">¹</a>.</p

Pathological assessment of SNpc and striatum in PQ- or MPTP-treated mice.

<p>Microscopic appearance of the SNpc (G-L) and striatum (A-F) in control (column 1), PQ- (column 2) or MPTP-treated (column 3) mice, 48 hours after dosing. Tyrosine hydroxylase (TH) immunostaining was decreased in the striatum (C) and SNpc (I) of the MPTP-treated animal but was unchanged in the PQ-treated mouse (B, H) compared to the control (A, G). Amino cupric silver (AmCuAg) staining was used to reveal degenerating neurons in the SNpc (J-L) and degenerating fibers in the striatum (D-F). There were no differences in AmCuAg staining in either the SNpc or striatum after PQ treatment compared to the control (D,J). AmCuAg staining was increased in the SNpc and striatum of the MPTP-treated mouse (F,L). The scale bar shown in Panel L represents 400 μm in panels A-C, G-I and 40 μm in panels D-F and J-L.</p

Mean histopathological severity scores in control, paraquat and MPTP-treated groups of C57BL/6J male mice.

<p>Mice were 16 weeks of age at the time of treatment initiation. Mice were administered 10 mg/kg/dose PQ·Cl₂ by ip injection, twice a week for 3 weeks and were sacrificed 8, 16, 24, 48, 96 or 168 hours after the last dose. Control mice were given the vehicle while MPTP-treated mice received four injections of MPTP (16 mg/kg/dose; expressed as free base) at 2-hour intervals, and then euthanized 48 hours after the final dose. Serial sections through the SNpc were evaluated qualitatively and the group mean severity grades are plotted. Grades 0 to 5 reflect increasing intensity of staining for Iba-1, AmCuGg, GFAP and decreased staining intensity of TH.</p

Appearance of microglia and astrocytes in SNpc and striatum of PQ- and MPTP-treated mice.

<p>Microscopic appearance of Iba-1-stained microglia (A-F) and GFAP-stained astroctyes (G-L) in the SNpc of mice 48 hours after PQ (column 2) or MPTP treatment (column 3) compared to a control (column 1). Iba-1 immunostaining of microglia was increased in the MPTP-treated mouse (C, F) but was not different in the PQ-treated mouse (B, E) compared to the control (A, D). Similarly, increased GFAP immunostaining of astrocytes was noted in the MPTP-treated mouse (I, L) but not in the PQ-treated mouse (H, K) compared to the control (G, J). Scale bar A-L 40 μm.</p