Zinc Oxide Nanoparticles (ZnO-NPs) Induce Cytotoxicity in the Zebrafish Olfactory Organs via Activating Oxidative Stress and Apoptosis at the Ultrastructure and Genetic Levels

Abstract

Nanotechnology has gained tremendous attention because of its crucial characteristics and wide biomedical applications. Although zinc oxide nanoparticles (ZnO-NPs) are involved in many industrial applications, researchers pay more attention to their toxic effects on living organisms. Since the olfactory epithelium is exposed to the external environment, it is considered the first organ affected by ZnO-NPs. Herein, we demonstrated the cytotoxic effect of ZnO-NPs on the olfactory organ of adult zebrafish after 60 days post-treatment. We opted for this period when fishes stop eating their diet from the aquarium, appear feeble, and cannot swim freely. Our study demonstrated that ZnO-NPs induced significant malformations of the olfactory rosettes at histological, ultrastructural, and genetic levels. At the ultrastructure level, the olfactory lamellae appeared collapsed, malformed, and twisted with signs of degeneration and loss of intercellular connections. In addition, ZnO-NPs harmed sensory receptor and ciliated cells, microvilli, rodlet, crypt, and Kappe cells, with hyper-activity of mucous secretion from goblet cells. At the genetic level, ZnO-NPs could activate the reactive oxygen species (ROS) synthesis expected by the down-regulation of mRNA expression for the antioxidant-related genes and up-regulation of DNA damage, cell growth arrest, and apoptosis. Interestingly, ZnO-NPs affected the odor sensation at 60 days post-treatment (60-dpt) more than at 30-dpt, severely damaging the olfactory epithelium and irreparably affecting the cellular repairing mechanisms. This induced a dramatically adverse effect on the cellular endoplasmic reticulum (ER), revealed by higher CHOP protein expression, that suppresses the antioxidant effect of Nrf2 and is followed by the induction of apoptosis via the up-regulation of Bax expression and down-regulation of Bcl-2 protein.

Keywords: apoptosis; olfactory epithelium; oxidative stress; zebrafish; zinc oxide nanoparticles (ZnO-NPs).

Figure 1

Gross morphology of the adult zebrafish (Danio rerio) olfactory rosette and main olfactory system structures: (A) Control zebrafish, (B) Lateral view of a dissected olfactory rosette (OR) of control fish (red arrow), (C) H&E histological assay of a longitudinal section of the whole control OR showing some lamellae; (D) ZnO-NPs treated zebrafish, (E) Lateral view of a dissected OR of treated fish (red arrow), and (F) H&E histological assay of a longitudinal section of the whole treated OR showing some lamellae.

Figure 2

H&E histological assay of (A) the control and (B) ZnO-NPs-treated zebrafish olfactory rosettes: (A(a)) horizontal section of the whole control olfactory rosette (OR), olfactory lamellae (OL), median raphe (MR); (A(b)) transverse section of the whole control OR, OL, MR, and central core (CC); (A(c)) magnified part of the OR showing CC, sensory epithelium (SE), ciliated receptor cells have darken spindle shape (CRC), cilia of the ciliated non-sensory cell (Ci), and goblet cell (GC); (A(d)) magnified part of the olfactory rosette showing supporting cell (SC) and basal cell (BC); (A(e)) magnified portion of the olfactory rosette showing CC; (A(f)) photomicrographs of the semi-thin section stained with toluidine blue of the olfactory lamella showing CC, SE, Ci, and GC (black arrow); (B(a)) horizontal section of the whole treated OR, collapsed OL, necrosis in MR; (B(b,c)) transverse section of the whole treated olfactory rosette showing swollen and detached olfactory epithelium (black arrow) and precipitation of ZnO-NPs in the olfactory epithelium and the central core (red arrow); (B(d)) magnified part of the olfactory rosette showing a large number of GC (black arrow); (B(e)) magnified portion of the olfactory rosette showing lamellar destruction (black arrow) and precipitation of ZnO-NPs (red arrow); (B(f)) photomicrographs of the semi-thin section of the olfactory lamella showing a large number of GC (black arrow) and precipitation of ZnO-NPs (red arrow). (C) Statistical analysis for three different sections from the OR of the control and treated group showing histopathological alterations after ZnO-NPs treatment. The results are the mean ± SEM.* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Scanning electron microscopy observations of the epithelial surface of (A) the control and (B) ZnO-NPs zebrafish olfactory rosette: (A(a)) scanning electron micrograph of the head of an adult zebrafish with two olfactory rosettes (ORs) (red arrows); (A(b)) olfactory rosette located in the nasal cavity, with olfactory lamellae (OL) arranged on both sides of the midline raphe (MR); (A(c)) higher magnification of the OL and the MR; (A(d,e)) sensory epithelium (SE) and indifferent epithelium (IE) are strictly separated; (A(f)) lamella is covered with ciliated non-sensory epithelium (CNE); (A(g)) cilia of the ciliated non-sensory cell (CNC); (A(h)) the surface of the sensory epithelium shows ciliated receptor cells (CRC) with olfactory knobs (OK), microvillous receptor cells (MV), and rod cells (RD); (B(a)) scanning electron micrograph of the head of an adult zebrafish with two treated OR (blue arrows); (B(b,c)) the olfactory rosette stunted appearance with preserved OL and the MR; (B(d)) numerous pores of mucous cells (MC), the IE without micro ridges; (B(e)) IE without micro-ridges; (B(f–h)) cilia of CNC fused with a decreased number.

Figure 4

Transmission electron micrographs of (A) the control and (B) ZnO-NPs-treated zebrafish olfactory epithelium: (A(a)) olfactory epithelium consisting of ciliated non-sensory cells (CNC) with cilia (Ci) and mitochondria (Mit); (A(b)) magnified part of ciliated non-sensory cells showing Mit; (A(c,d)) cross-section of cilia exhibits nine pairs of outer microtubules (Mi) and two central ones (red arrow), basal body (bb); (A(e)) magnified view of ciliated receptor cells (CRC) showed an olfactory knob (Ok) bearing cilia (Ci). (A(f)) magnified view of microvillous receptor cell (MV), which showed an Ok bearing short microvillar processes (Mi) on its apical surface and Mit; (A(g)) magnified view of rod cell (RD) shows parallel oriented microtubules (red arrows) in rod-like cilia (R) and Mit. (A(h)) the rodlet cell (Rt) is identified by the thick cuticle (cu) and its typical rodlets (red arrows) and nucleus (N); (B(a–d)) olfactory epithelium consisting of ciliated non-sensory cells with decreased number of Ci and Mit swelling and vacuolation (blue arrow); (B(e–g)) the cilia, microvilli, and rods of all olfactory neurons injured mitochondria swollen and vacuolated (blue arrow); (B(h)) rodlet cell with hypertrophy nucleus (blue star) and detached apical rodlets (blue arrow).

Figure 5

Transmission electron micrographs of the degenerated sensory cells of (A) the control and (B) ZnO-NPs-treated zebrafish olfactory epithelium: (A(a)) crypt cell (Cc) with in-sunk cilia, the nucleus (N) in the lower portion of the cell, supporting cell (SC) surrounding the crypt cell and mitochondria (Mit); (A(b)) higher magnification of the upper portion of a crypt cell, (Cc) showing cilia (Ci) and microtubules (Mi). (A(c)) the Kappe cells (KP), the nucleus (N) in the basal part of the cell, micro-ridges upper part (red arrows), and mitochondria (Mit); (A(d)) pear-shaped cell (Pr), the cytoplasm with mitochondria (Mit) and ribosomes (red arrows); (A(e,f)) basal cells (BC, red arrow) with N; (A(g,h)) goblet cells (GC); (B(a,b)) crypt cell (Cc) showing the disappearance of cilia, elongated mitochondria (blue star), and the surrounding supporting cells (SC) thinned out; (B(c,d)) the Kappe and pear-shaped cells that have lost their distinct shape (blue star); (B(e,f)) basal cells (BC) increased in size, and with the nucleus (N) enlarged; (B(g,h)) hyper-activated goblet cells (GC) with retention of a large amount of ZnO-NPs (blue star).

Figure 6

ZnO-NPs-induced cellular apoptosis via oxidative stress and DNA damage at the genetic level: (A) mRNA expression evaluated by qRT-PCR for antioxidant-related genes; (B) mRNA expression evaluated by qRT-PCR for DNA damage-related genes; (C) mRNA expression evaluated by qRT-PCR for apoptosis-related genes. The results are the mean ± SEM.* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Apoptotic pathway induction by ZnO-NPs via oxidative stress induction: (A) immunoblots showing Nrf2, CHOP, Bax, and Bcl-2 proteins of control, 30-dpt, and 60-dpt (β-actin was used for normalization and relative protein levels were quantified using NIH software Image J), (B–E) protein expression revealing the ratio analysis of Nrf2/βactin, CHOP/βactin, BAX/β actin, Bcl-2/βactin quantified by the Image J 1.51k; Java 1.6.0_24 (64-Bit). The results are the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.