Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Abstract

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.

Fig. 1

Representative confocal images obtained from haemocytes loaded with zinc-specific fluorochrome FluoZin-3 AM, not exposed to Zn ions (control) and treated for 3 h with 0.35, and 1.7 mM of Zn²⁺. The histogram represents the average values (±SE) of free Zn ion concentrations (nM) in the fruit fly’s haemocytes calculated on the basis of fluorescence intensity of FluoZin-3. **Statistically significant differences between experimental groups and control (p < 0.01, N = 3)

Fig. 2

The effect of Zn treatment (0.35 and 1.7 mM) on D. melanogaster MtnA and MtnB gene expression in larval haemocytes quantified as mRNA levels using real-time PCR. Data are shown as RQ index calculated from ∆∆C _T method (mean ± SE) normalized to the control. **Statistically significant differences between experimental groups and control (p < 0.01, N = 6)

Fig. 3

The activity of caspases DrICE, Dronc and Dredd in D. melanogaster haemocytes in the presence of 0.35 and 1.7 mM of Zn ions in comparison to the control. The results (means ± SE) are normalized to the level of caspase activity measured in the control cells. * and **Statistically significant differences between experimental groups and control for p < 0.05 and p < 0.01, respectively (N = 3)

Fig. 4

The results of DIAP1 expression measurements in D. melanogaster haemocytes treated for 3 h with 0.35 and 1.7 mM of zinc ions and in the control, Zn-untreated cells. Representative confocal images obtained from experimental and control groups of haemocytes. The intensity of red fluorescence exhibits the level of DIAP1 protein present in haemocyte cytoplasm. All micrographs were taken at the same imaging parameters using C-Apochromat 40×/1.2 W corr objective. The histogram represents mean values (±SE) of Cy-3 fluorescence bound to DIAP1 proteins after immunolabelling with anti-DIAP1 serum (see “Materials and methods”) in the fruit fly’s haemocytes. **Statistically significant differences between experimental groups and control (p < 0.01, N = 3)

Fig. 5

Changes in mitochondrial membrane potential in D. melanogaster haemocytes after their treatment with Zn ions. Haemocytes were stained with JC-1 indicator and intensity of red fluorescence was analyzed fluorometrically. Living haemocytes showed strong accumulation of JC-1 indicating intact mitochondria. After zinc treatment, disruption of mitochondrial membrane potential was observed leading to the significant decrease of JC-1 red fluorescence intensity. Data were normalized to the control samples and are shown as mean ± SE. Asterisk indicates statistically significant differences between experimental and control samples (p < 0.05, N = 3)