Morais F et al. (2024), Influence of polystyrene nanoplastics on the to...

XB-ART-60877

Sci Total Environ 2024 Aug 11;:175375. doi: 10.1016/j.scitotenv.2024.175375.

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Influence of polystyrene nanoplastics on the toxicity of haloperidol to amphibians: An in vivo and in vitro approach.

Morais F , Pires V , Almeida M , Martins MA , Oliveira M , Lopes I .

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Chemical pollution is a major driver for the current worldwide crisis of amphibian decline. The present study aimed to assess the influence of polystyrene nanoplastics (PS-NPLs) on the toxicity of haloperidol to aquatic life stages of amphibians, by using in vivo (tadpoles of Xenopus laevis and Pelophylax perezi) and in vitro (A6 and XTC-2 cell lines of X. laevis) biological models. Tadpoles of both species were exposed, for 96 h, to haloperidol: 0.404 to 2.05 mg l-1 (X. laevis) or 0.404 to 3.07 mg L-1 (P. perezi). The most sensitive species to haloperidol (X. laevis) was exposed to haloperidol's LC50,96h combined with two PS-NPLs concentrations (0.01 mg L-1 or 10 mg L-1); the following endpoints were monitored: mortality, malformations, body lengths and weight. In vitro cytotoxicity was assessed by exposing the two cell lines, for 72 h, to: haloperidol (0.195 to 100 mg L-1) alone and combined with 0.01 mg L-1 or 10 mg L-1 of PS-NPLs. Xenopus laevis tadpoles revealed a higher lethal and sublethal sensitivity to haloperidol than those of P. perezi, with LC50,96h of 1.45 and 2.20 mg L-1. In vitro assays revealed that A6 cell line is more sensitive haloperidol than XTC-2: LC50,72h of 13.2 mg L-1 and 5.92 mg L-1, respectively. Results also suggested a higher sensitivity of in vivo models when compared to in vitro biological. Overall, PS-NPLs did not influence haloperidol's toxicity for in vivo and in vitro biological models, except for a reduction on the incidence of malformations while increasing the lethal toxicity (at the lowest concentration) in tadpoles. These opposite interaction patterns highlight the need for a deeper comprehension of NPLs and pharmaceuticals interactions. Results suggest a low risk of haloperidol for anuran tadpoles, though in the presence of PS-NPLs the risk may be increased.

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Species referenced: Xenopus laevis

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	Graphical abstract
	Fig. 1. Proportion of alive tadpoles of Xenopus laevis and Pelophylax perezi exhibiting malformations after being exposed, for a period of 96 h, to several haloperidol concentrations. Data shown for X. laevis exposed to 2.05 mg L−1, corresponds to one alive tadpole. Ctr: corresponds to the negative control with FETAX medium; Ctr-Sol: corresponds to the control solvent with 0.01 % of DMSO. He-hemorrhage, Bt-bent tail, E-Oedema, Hpe-hypopigmentation, Shb-slow heart-beat. * Significant differences from the control (Chi-squared test: p < 0.05).
	Fig. 2. Average weight of tadpoles of Xenopus laevis and Pelophylax perezi, after being exposed, for a period of 96 h, to several haloperidol concentrations. Error bars represent standard deviation. * Indicates significant differences relatively to the control (Dunnett's test: p < 0.0001). Ctr: corresponds to the negative control with FETAX medium; Ctr-Sol: corresponds to the control solvent with 0.01 % of DMSO. For X. laevis, results presented for concentration 2.05 mg L−1 correspond to one alive tadpole.
	Fig. 3. Average length of total body (TBL), snout-to-vent (SVL) and tail (TL) of tadpoles of Xenopus laevis and Pelophylax perezi, after being exposed, for 96 h, to several haloperidol concentrations. Error bars represent standard deviation. * Indicates significant differences relatively to the control (Dunnett's test: p < 0.0001). Ctr: corresponds to the negative control with FETAX medium; Ctr-Sol: corresponds to the control solvent with 0.01 % of DMSO. For X. laevis, results presented for concentration 2.05 mg L−1 correspond to one alive tadpole.
	Fig. 4. Viability fitted curves (4P) of XTC-2 (left) and A6 (right) cell lines when exposed to haloperidol concentrations in 3 different time-points, 24 h, 48 h and 72 h.
	Fig. 5. Percentage of mortality (average) and malformations (total %) for Xenopus laevis tadpoles after being exposed, for 96 h, to haloperidol and PS-NPLs, as single and in mixtures: HPS-NPLs-10 mg L−1 of PS nanoplastics, LPS-NPLs-10 μg L−1 of PS nanoplastics, HAL-LC50,96h of haloperidol, HAL + HNPLs- LC50,96h of haloperidol +10 mg L−1 of PS nanoplastics, HAL + LNPLs- LC50,96h of haloperidol +10 μg L−1 of PS nanolpastics. In the graph of mortality: error bars represent standard deviation and letters (a, b) indicates homogenous groups (Chi-squared test: p < 0.05). He-Hemorrhage, E-Edema, Hpe- hypopigmentation.
	Fig. 6. Percentage of Xenopus laevis tadpoles at the development stages NF47 or NF48, after a period of 96 h of exposure to haloperidol and PS-NPLs, as single and in mixtures: HPS-NPLs-10 mg L−1 of PS nanoplastics, LPS-NPLs-10 μg L−1 of PS nanoplastics, HAL-LC50,96h of haloperidol, HAL + HPNPLs- LC50,96h of haloperidol +10 mg L−1 of PS nanoplastics, HAL + LNPLs- LC50,96h of haloperidol +10 μg L−1 of PS nanolpastics.
	Fig. 7. Average weight and body lengths (total body-TBL, snout-to-vent-SVL, tail-TL) of tadpoles of Xenopus laevis, after being exposed for 96 h to haloperidol (HAL) and PS-NPLs, alone and in combined mixtures: HPS-NPLs-10 mg L−1 of PS nanoplastics, LPS-NPs-10 μg L−1 of PS nanoplastics, HAL-LC50,96h of haloperidol, HAL + HNPLs- LC50,96h of haloperidol +10 mg L−1 of PS nanoplastics, HAL + LNPLs- LC50,96h of haloperidol +10 μg L−1 of PS nanoplastics. Letters (a, b, c) represent homogenous groups within each endpoint (p < 0.0001).
	Fig. 8. Viability fitted curves of XTC-2 (left) and A6 (right) cell lines when exposed to a combination of haloperidol (HAL) with high concentration of PS-NLPs (HAL + HNPLs) of 10 mg L−1 and a low concentration of PS-NPs (HAL + LNPLs) of 10 μg L−1 in 3 different time-points, 24 h, 48 h and 72 h. Data presented as fitted curves (4P).