Early Exposure to Environmental Pollutants: Imidacloprid Potentiates Cadmium Toxicity on Zebrafish Retinal Cells Death

Abstract

In the present study, we analyzed the combination of non-toxic concentrations per se, of Cd and a pesticide the imidacloprid (IMI) (10 and 50 μM for Cd and 195 μM for IMI), to highlight early developmental toxicity and possible damage to retinal cells. Co-exposure to Cd and IMI showed a toxic effect in zebrafish larval development, with lowered degrees of survival and hatching, and in some cases the induction of structural alterations and edema. In addition, co-exposure to 50 and 195 μM, respectively, for Cd and IMI, also showed increased apoptosis in eye cells, accompanied by up regulation of genes associated with antioxidant markers (cat, sod1, nrf2 and ho-1). Thus, the present study aims to highlight how the presence of multiple contaminants, even at low concentrations, can be a risk factor in a model of zebrafish (Danio rerio). The presence of other contaminants, such as IMI, can cause an enhancement of the toxic action of Cd on morphological changes in the early life stage of zebrafish, but more importantly disrupt the normal development of the retina, eventually triggering apoptosis.

Keywords: apoptosis; environment contaminant; pesticides.

Figure 1

The effects of Cd and IMI on total ell death in zebrafish embryos. (A) Control; (B) Cd 10 μM; (C) Cd 50 μM; (D) IMI 195 μM; (E) Cd 10 μM + IMI 195 μM; (F) Cd 50 μM + IMI 195 μM; Cell death death (G). At 120 hpf, Cd 50 μM, IMI 195 μM and Cd 50 μM + IMI 195 μM incubation, the levels of total cell death were observed and photographed by a fluorescence microscope after staining with acridine orange. Values are expressed as means ± SEM of three independent experiment data; *** at p < 0.001 against CTRL.

Figure 2

Detection of Cd and IMI exposure indued changes in the cell death within zebrafish eyes. Cell apoptosis within the eyes of live zebrafish embryos at 120 hpf determined using acridine orange (AO) staining. (A) Control; (B) Cd 10 μM; (C) Cd 50 μM; (D) IMI 195 μM; (E) Cd 10 μM + IMI 195 μM; (F) Cd 50 μM + IMI 195 μM; Cell death signals were indicated by green fluorescent spot on a indicated by the blank square (F); (G) Effect Cd and IMI on eye diameter at 120 hpf. Values are expressed as means ± SEM of three independent experiment data; *** at p < 0.001 against CTRL.

Figure 3

TUNEL assays indicated an abnormal apoptotic pattern. TUNEL-positive apoptotic cells (white arrow) in zebrafish embryos treated with Cd and IMI at 120 hpf. (A) Control; (B) Cd 10 μM; (C) Cd 50 μM; (D) IMI 195 μM; (E) Cd 10 μM + IMI 195 μM; (F) Cd 50 μM + IMI 195 μM.

Figure 4

Effects of Cd and IMI exposure on the mRNA levels of stress oxidative pathway sod1, cat, nrf2 and ho-1 in larval zebrafish. Values are expressed as means ± SEM of three independent experiment data; *** at p < 0.001 against CTRL.

Figure 5

Effects of Cd and IMI exposure in ROS formation (A) and MDA (B) in zebrafish. Values are expressed as means ± SEM of three independent experiment data; * at p < 0.05 against CTRL *** at p < 0.001 against CTRL.