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First published online August 23, 2004
Journal of Experimental Biology 207, 3369-3380 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01152
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Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition

I. M. Sokolova1,{dagger},*, S. Evans2 and F. M. Hughes1,{dagger}

1 Biology Department, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223, USA
2 Johnson C. Smith University, 100 Beatties Ford Road, Charlotte, NC 28216, USA



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  		Fig. 1. Morphology of control oyster hemocytes (A,B) and presumptive apoptotic hemocytes exposed to 50 µmol l-1 of cadmium (C,D). Horizontal bars correspond to 10 µm. Note extensive blebbing in cadmium-exposed hemocytes (C,D).

 


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  		Fig. 2. Induction of apoptosis and necrosis by cadmium exposure in oyster hemocytes. (A) Representative dot plots for annexin V-FITC and propidium iodide (PI) staining in hemocytes exposed to vehicle (control) or varying concentrations of cadmium for 72 h. Live cells appear in the bottom left quadrant of the dot plot and have low FITC and PI fluorescence. Apoptotic cells in the bottom right quadrant are characterized by low PI fluorescence, indicating integrity of the plasma membrane, but high FITC fluorescence due to the translocation of phosphatidylserine into the outer leaflet of the plasma membrane. Necrotic cells have high PI and FITC fluorescence and are found in the upper right quadrant of the dot plot. (B,C) Quantitative graph of the data shown in A indicating the changes in the proportion of apoptotic (B) and necrotic (C) cells in the hemocyte population after 72 h of exposure to different cadmium concentrations. Vertical bars represent S.E.M. Filled circles correspond to the values that were significantly different from the respective control (0 µmol l-1 Cd2+; P<0.05; N=5-8). The levels of apoptosis and necrosis in control hemocytes after 72 h of culture were not significantly different from those in freshly isolated oyster blood cells (12.0±1.70 and 0.9±0.34% of apoptosis and necrosis, respectively, N=10, P>0.05), indicating that culture conditions used in these experiments supported normal viability of oyster hemocytes.

 


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  		Fig. 3. Changes in mitochondrial membrane potential ({Delta}{Psi}m) in isolated oyster hemocytes exposed to different cadmium concentrations for 72 h as measured by TMRM staining. (A) Representative distributions of the TMRM fluorescence intensity of control and cadmium-exposed cells before or after treatment with a mitochondrial uncoupler (CCCP). (B) Quantitative graph of the data shown in A indicating the average TMRM fluorescence intensity in control cells and cells exposed to different cadmium concentrations before (-CCCP) and after (+CCCP) exposure to the mitochondrial uncoupler CCCP. The striped bar represents TMRM fluorescence in control hemocytes without digitonin treatment. Vertical bars represent S.E.M. *Values are significantly different from the control (0 µmol l-1 Cd2+); {dagger}values significantly different from the fluorescence intensity of the respective cell populations in the absence of CCCP (P<0.05; N=6-8).

 


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  		Fig. 4. Caspase-3 activity in freshly isolated oyster hemocytes (Fresh) and hemocytes incubated for 72 h with vehicle, 50 or 200 µmol l-1 of cadmium. Cyt c: activation of caspase-3 from vehicle-treated hemocytes with cytochrome c and dATP. Apoptotic thymocytes: caspase-3 activity in growth-factor-deprived murine thymocytes used as a positive control. Vertical bars represent S.E.M. Values that are not significantly different from each other are denoted by the same letters (P<0.05; N=5-6).

 


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  		Fig. 5. Changes in the intracellular ATP concentration in oyster hemocytes exposed to different cadmium concentrations for 72 h. Vertical bars represent S.E.M. Asterisks denote values that are significantly different from the control (P<0.05; N=7-13).

 


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  		Fig. 6. Effects of cadmium on respiration rate and coupling of isolated oyster mitochondria. State 3, ADP-stimulated respiration; state 4+, respiration in the presence of oligomycin (indicative of proton leak); RCR, respiratory control ratio of state 3 over state 4+ respiration. Vertical bars represent S.E.M. Asterisks, daggers and filled circles denote values of state 3, state 4+ respiration and RCR, respectively, which are significantly different from the control (P<0.05; N=5-6).

 


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  		Fig. 7. Effects of cadmium on mitochondrial volume (A) and membrane potential (B) in isolated oyster mitochondria. (A) Mitochondrial swelling was measured as a decrease of absorbance at 520 nm. {Delta}Abs520, difference in absorbance at 520 nm between control mitochondria and those incubated with different concentrations of cadmium. This method only gives qualitative data about the mitochondrial volume change; positive values of {Delta}Abs520 indicate mitochondrial swelling, while negative values indicate mitochondrial contraction. (B) Mitochondrial membrane potential was measured as a ratio of fluorescence of mitochondria incubated with TMRM at excitation wavelengths of 573 or 546 nm and an emission wavelength of 590 nm. Ratio of fluorescence at 573 nm over 546 nm excitation wavelengths is linearly proportional to membrane potential ({Delta}{Psi}m). Vertical bars represent S.E.M. Asterisks denote values that are significantly different from the control (P<0.05; N=5-6).

 

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© The Company of Biologists Ltd 2004