Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

Abstract

Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway.

Fig 1. Cd induced apoptosis in gill cells of freshwater crab S. henanense.

Crabs were treated with 58 mg•L^-1 CdCl₂ for 48 h and cell apoptosis was assessed. (A) The effects of Cd on the morphology of nuclei. (a) (×8000) normal nuclei in untreated control group. (b) (×8000) abnormal nuclei with apoptotic characteristics in the Cd-treated group. (B) DNA fragmentation characteristics of gill cells by Cd. Mr: DNA marker, Con: control (untreated gill cells), Cd: Cd-treated gill cells. (C) The effects of Cd on the activities of caspases-3, -8 and -9. The mean expression in each treated group is shown as a fold increase compared to the mean expression in the control, which had been ascribed an arbitrary value of 1. *P<0.05, **P<0.01 difference vs. control group.

Fig 2. Effects of acute Cd exposure on [Ca²⁺]i in gill cells of S. henanense.

Cells were loaded with 5 μM Fluo-3/AM for 30 min at 37°C in the dark, then treated with 5.8 mg•L^-1 CdCl₂ for the different times, followed by the images recording under a laser confocal microscope (LSM 510 META, Leica, Bensheim, Germany).

Fig 3. Effects of Cd exposure on CaM content and Ca²⁺-ATPase activity in gill cells.

Results are presented as mean ± SE; n = 3. *P<0.05, **P<0.01 difference vs. control group.

Fig 4. Effects of pretreatment with EGTA on DNA fragmentation induced by Cd.

DNA fragmentation was assayed in agarose gel electrophoresis. Crabs were pretreated with or without 5 mM EGTA for 4 h, and then exposed to 58 mg•L^-1 Cd for 48 h. The letters on the lanes represent: Con = H₂O (4 h) + H₂O (48 h); Cd = H₂O (4 h) + 58 mg•L^-1 CdCl₂ (48 h); E = 5 mM EGTA (4 h) + H₂O (48 h); and, E + Cd = 5 mM EGTA (4 h) + 58 mg•L^-1 CdCl₂ (48 h).

Fig 5. Effects of EGTA pretreatment on Cd-induced morphological variation of epithelial cells in the gill.

(A) (×10000) normal epithelial cell with nucleus and a large of cytoplasmic organelles. (B) (×10000) apoptotic epithelial cell induced by 58 mg•L^-1 CdCl₂ with apoptotic characteristics, such as chromatin condensation and extremely irregular nuclear membrane in nucleus. (C) (×8000) epithelial cell treated by 5 mM EGTA alone; (D) (×6000) epithelial cell treated by 5 mM EGTA for 4 h, then exposed to Cd for 48 without apoptotic characteristics.

Fig 6. Effects of pretreatment with EGTA on the activities of caspase enzymes in the gill of crabs exposed to acute Cd treatment.

The mean expression in each treated group is shown as a fold increase compared to the mean expression in the control, which had been ascribed an arbitrary value of 1. Using one-way analysis of variance and on comparing with the control, significance is shown by *P<0.05, **P<0.01; on comparing with Cd treatment group, ^# P<0.05, ^## P<0.01.