Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis

Abstract

Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic.

Keywords: Atropine; apoptosis; cat corneal endothelial cells; cytotoxicity; human corneal endothelial cells; mitochondrion.

Figure 1

Growth status and morphological changes of atropine-treated HCE cells. Cultured HCE cells were treated with the indicated concentration and exposure time of atropine, and their growth and morphology were monitored by light microscopy. One representative photograph from three independent experiments was shown

Figure 2

Cell viability decline and cell cycle arrest of atropine-treated HCE cells. Cultured HCE cells were treated with the indicated concentration and exposure time of atropine. (a) MTT assay. The cell viability of atropine-treated HCE cells in each group was expressed as percentage (mean ± SD) of 490 nm absorbance compared to its corresponding control (n = 3). b, P < .01 vs control. (b) FCM by PI staining. One representative image from three independent experiments was shown. The cell number of HCE cells in each phase of the cell cycle was expressed as percentage of the total number of cells

Figure 3

In vivo examination of 40 g/L atropine-exposed cat eyes. (a) Endothelial cell density (ECD) of CCE cells. (b) Central corneal thickness (CCT) of cat corneas. (c) Intraocular pressure (IOP) of cat eyes. Data in (a), (b), and (c) were all expressed as mean ± SD (n = 4). a, P < .05, b P < .01 vs normal control. (d) Ex vivo microscopic photographs of CCE cells 30 days after exposed to atropine. One representative photograph from four cats was shown. The swollen cells in alizarin red staining and SEM images were indicated by arrow heads, and swollen mitochondria in TEM images were indicated by arrows. SEM: scanning electron microscopy; TEM: transmission electron microscopy

Figure 4

PS externalization and plasma membrane permeability elevation of atropine-treated HCE cells. Cultured HCE cells were treated with the indicated concentration and exposure time of atropine and their plasma membrane abnormality were assayed. (a) FCM by Annexin V/PI staining. Annexin V⁺ cells (PS-externalized cells) were identified as apoptotic cells. The cell number of HCE cells in each group was expressed as percentage (mean ± SD) of the total number of cells (n = 3). a, P < .05, b P < .01 vs control. (b) AO/EB double staining. The apoptotic ratio was calculated as percentage (mean ± SD) of the total number of cells based on the permeability elevation of plasma membrane of HCE cells (n = 3). a, P < .05, b P < .01 vs control

Figure 5

DNA fragmentation and ultrastructural abnormity of atropine-treated HCE cells. Cultured HCE cells were treated with the indicated concentration and exposure time of atropine. (a) DNA electrophoresis. DNA isolated from HCE cells was electrophoresed in 1% (w/v) agarose gel, and dispersed DNA ladders were shown. (b) TEM photographs. One representative photograph from three independent experiments was shown. N: nucleus; RER: rough endoplasmic reticulum; chn: condensed chromatin; m: swollen mitochondrion; mv: microvillus; v: vacuole; *: apoptotic body

Figure 6

Caspase activation and MTP disruption in atropine-treated HCE cells. Cultured HCE cells were treated with 2.5 g/L atropine for the indicated exposure time. (a) ELISA detection. Caspase activation was measured by ELISA using monoclonal antibodies against the active form of caspase-2, -3, and -9. The activation ratio of each group was expressed as percentage (mean ± SD) compared to its corresponding control based on the 450 nm absorbance (n = 3). a, P < .05; b, P < .01 vs control. (b) FCM using JC-1 staining. JC-1 in mitochondria with MTP depolarized maintains monomers in green fluorescence, while that in mitochondria with normal MTP incorporates into aggregates in red fluorescence. The number of JC-1 positive cells (MTP-disrupted cells) in each group was expressed as percentage (mean ± SD) of the total number of cells (n = 3). b, P < .01 vs control

Figure 7

Altered expression of Bcl-2 family proteins and cytoplasmic translocation of Cyt. c, and AIF in atropine-treated HCE cells. Cultured HCE cells were treated with 2.5 g/L atropine for the indicated exposure time, and the expression level of Bcl-2 family proteins and mitochondrion release of apoptotic proteins were examined by western blotting. (a) Western blot images. (b) Densitometry analyses. The relative level of protein expression was expressed as percentage (mean ± SD) of protein band density compared to the internal control of β-actin (n = 3). b, P < .01 vs control