Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce locomotor deficits and dopaminergic degeneration in Caenorhabditis elegans

Abstract

Organophosphate flame retardants (PFRs) are a new class of flame retardants. The health risks of PFRs have received attention recently. However, little is known about the potential toxicity of PFRs on the nervous system. Herein, we evaluated the neurotoxic effects of two typical PFRs, tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP), using Caenorhabditis elegans. Median lethal concentrations of chronic exposure (3 d) were 1578 and 857 mg L^-1 for TCEP and TCPP, respectively. The sublethal dose of TCEP or TCPP significantly inhibited the body length and reduced the lifespans of nematodes. 500 mg L^-1 and above of TCEP/TCPP led to a significant decline in the locomotor frequency of body bending and head thrashing. Furthermore, their exposure reduced the crawling speed and the frequency of bending oscillation of nematodes. This indicates that TCEP/TCPP induces locomotor deficits, along with Parkinsonian-like movement impairment including bradykinesia and hypokinesia. Using transgenic worms, we found that TCEP/TCPP could induce down-expression of P _dat-1 and resulted in the degeneration of dopaminergic neurons, especially PDE neurons. Moreover, TCEP/TCPP induced over-expression of unc-54, which indicates the aggregation of α-synuclein in the process of degeneration. These findings suggest the neurotoxicity risks of organophosphorus flame retardants, which are associated with the locomotor deficits and dopaminergic degeneration.

Fig. 1. Survival percentages of C. elegans after being exposed to TCEP (A) and TCPP (B) for 1–6 d. Data are expressed as means ± SD of four separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, when compared to the control.

Fig. 2. Effects of TCEP and TCPP on body lengths and lifespans in C. elegans. Body lengths (A1, A2), the survival curves (B1, B2) and mean lifespans (C1, C2) of C. elegans after respectively being exposed to TCEP and TCPP for 3 d. Data are means ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control.

Fig. 3. Effects of TCEP and TCPP on local locomotor behavior in C. elegans. Body bends (A1, A2), head thrashes (B1, B2) and pharynx pumping (C1, C2) of C. elegans after respectively being exposed to TCEP and TCPP for 3 d. Data are means ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control.

Fig. 4. Effects of TCEP and TCPP on the crawling movement in C. elegans. (A1, A2) 60 s crawling track line of nematodes after respectively being exposed to TCEP (A1) and TCPP (A2) with different concentrations for 3 d; (B1, B2) mean crawling speed of nematodes after respectively being exposed to TCEP (B1) or TCPP (B2); (C1, C2) distribution of the real-time crawling speed of C. elegans, after respectively being exposed to TCEP (B1) and TCPP (B2). Data are means ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control.

Fig. 5. Changes of bending angles of crawling locomotor of C. elegans after being exposed to TCEP and TCPP for 3 d. (A1 and A2) 10 s real-time recording of the bending degree in C. elegans after TCEP (A1) or TCPP (A2) exposure; (B1 and B2) the frequency of mid-body bending of worms after exposure to TCEP (B1) or TCPP (B2). Data are means ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control. The width of the bin is 10 s in A1 and A2.

Fig. 6. Effects of TCEP and TCPP exposure on the GFP expression pattern in dopaminergic neurons of the transgenic C. elegans strain BZ555. (A) The diagrammatic figure of dopaminergic neurons in C. elegans, which include four CEP (cephalic neurons), two ADE (anterior deirid neurons) and two PDE (posterior deirid neurons). (B) In the control nematodes, dopaminergic neurons are visualized by the translational expression of the GFP driven by the promoter of the dopamine transporter (P_DAT-1::GFP). (C–F) Fluorescence images show dopaminergic neurons in worms after being exposed to 750, 1000 mg L^–1 of TCEP (C, E), and 500,750 mg L^–1 of TCPP (D, F) for 3 d, respectively. The white box shows the amplified tail region of worms. Red arrows show the loss of neuronal soma or neurite in dopaminergic neurons. (G, H) The fluorescence intensity of the GFP expression pattern in dopaminergic neurons after exposure to TCEP (G) and TCPP (H). Data are means ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control. Bar = 200 μm.

Fig. 7. Effects of TCEP and TCPP exposure on the α-synuclein protein (labeled by using the yellow fluorescent protein) in the transgenic C. elegans strain NL5901. (A) Fluorescence images show the α-synuclein protein in the muscles of control worms. (B–E) Fluorescence images show the α-synuclein protein of nematodes after respectively being exposed to 750, 1000 mg L^–1 of TCEP (B, D), and 500, 750 mg L^–1 of TCPP (C, D) for 3 d. (F, G) The fluorescence intensity of the yellow fluorescent protein shows α-synuclein expression in the nematodes after being exposed to TCEP (F) or TCPP (G). *p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control. Bar = 200 μm.