Cytotoxicity and Oxidative Stress Responses of Imidacloprid and Glyphosate in Human Prostate Epithelial WPM-Y.1 Cell Line

Abstract

Insecticide imidacloprid and herbicide glyphosate have a broad spectrum of applicable use in the agricultural sector of Egypt. Their ability to induce in vitro cytotoxic and oxidative stress on normal human cells (prostate epithelial WPM-Y.1 cell line) was evaluated with the methyl tetrazolium test (MTT) and histopathological investigation. Cell viability was evaluated with an MTT test for 24 h. The median inhibition concentration (IC₅₀) values were 0.023 and 0.025 mM for imidacloprid and glyphosate, respectively. Sublethal concentrations: 1/10 and 1/50 of IC₅₀ and IC₅₀ levels significantly induced an increase in the lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) level compared with the untreated cells. Rapid decrease in the glutathione (GSH) content and glutathione-S-transferase (GST) activity was induced. Significant increases were recorded in activities of catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), respectively, compared with the control group. Transmission electron microscopic (TEM) investigation showed significant defects in the cells following pesticide treatments for 24 h. Therefore, it is concluded that imidacloprid and glyphosate are very toxic in vitro assays and able to induce apoptotic effects as well as oxidative stress. So, these findings provide a scenario of multibiomarkers to achieve the imposed risks of pesticides at low doses.

Figure 1

Acute toxic effect (cell death %) of the examined pesticides: (a) imidacloprid and (b) glyphosate on WPM-Y.1 cell line after 24 h exposure estimated as IC₅₀.

Figure 2

Pesticides-induced morphological changes in WPM-Y.1 cells. WPM-Y.1 cells were seeded in 12-well plates and allowed to grow for 20 h. Thereafter, cells were exposed to the IC₅₀ level, 0.023 and 0.025 mM of imidacloprid and glyphosate, respectively, for 24 h. Significant alterations were induced in cell membranes and destructed their cytoplasm and microvilli which appeared as desorbed cells. The morphological changes were analyzed using the LABOMED inverted microscope, USA. Images selected were performed with a magnification of 20x.

Figure 3

(a) Lactate dehydrogenase (LDH) activity (U L⁻¹) and (b) malondialdehyde (MDA) level (mM g⁻¹ tissue) in WPM-Y.1 cells exposed to different concentrations (mM) of imidacloprid and glyphosate. Each value indicates the mean of 3 replicates ± SE.

Figure 4

(a) Glutathione content (GSH), (b) catalase (CAT), (c) glutathione-S-transferase (GST), (d) glutathione peroxidase (GPx), and (e) glutathione reductase (GR) activities, respectively, in WPM-Y.1 cells exposed to different concentrations (mM) of imidacloprid and glyphosate. Each value represents the mean of 3 replicates ± SE. The same letters indicate no significant difference at the 0.05 level.

Figure 5

(a) Electron micrograph illustrates the section of untreated WMP-Y. 1 cells with regular nuclear membrane (head arrow). Smooth endoplasmic reticulum (SER), golgi bodies (G), and mitochondria (M) with light dense cristae. Highly distributed lysosomes (Ly). Moreover, significant double nuclei (Nu) were observed (F₄G₁-OsO₄ fixed-uranyl acetate lead citrate stained preparation, 4000x). (b) High magnification showing normal distribution of chromatin and normal distribution of spherical mitochondrial organelle around the nucleus. Highly and distribution of golgi bodies and some vacuoles (8000x).

Figure 6

(a) Electron micrograph of imidacloprid-treated cells (0.0023 mM) illustrates destructed cell's organelles and no cellular membrane appeared (F₄G₁-OsO₄ fixed-uranyl acetate lead citrate stained preparation, 4000x). (b) High magnification at 8000x shows nuclear-destructed components.

Figure 7

(a) Electron micrograph of glyphosate-treated cells (0.0025 mM) illustrates irregular nuclear membrane with highly migrated chromatin (head arrow) and lack of nuclei (Nu). Rough endoplasmic reticulum (RER), high distributed vacuoles (V), and some autolysomes (Ly) were noted (F₄G₁-OsO₄ fixed-uranyl acetate lead citrate stained preparation, 4000x). (b) High magnification at 8000x shows compacted organelles and a lack of nuclei.