DOSE-DEPENDENT INDUCTION OF APOPTOSIS OR NECROSIS IN HUMAN-CELLS BY ORGANOTIN COMPOUNDS

In the present study, the mode of cell death induced by highly toxic t rialkylated tin compounds has been evaluated. Treatment of undifferent iated HL-60 cells with submicromolar to micromolar concentrations of t ri-n-butyl-tin (TBT) led only to a slight decrease in cell viability m easured with trypan blue exclusion. Nevertheless, cell membrane blebbi ng was observed by means of light microscopy and condensation of nucle ar chromatin and formation of apoptotic bodies was demonstrated in Hoe chst 33342 stained cells. The nuclear chromatin condensation was assoc iated with an extensive DNA fragmentation. Visualized by agarose gel e lectrophoresis, genomic DNA appeared as a characteristic ladder-like p attern of DNA fragments which is the biochemical hallmark of apoptosis . The typical internucleosomal DNA digestion was concentration-depende nt and began within 2 to 3 h of incubation. During the incubation peri od a persistent and steady elevation of intracellular free calcium con centration ([Ca2+](i)) could be detected. Furthermore, the chromatin c ondensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc -sensitive and calcium-dependent endogenous endonuclease. Higher conce ntrations of tributyltin (greater than or equal to 5 mu mol/L TBT) led within hours to a cell killing with degenerative changes indicative o f necrosis, demonstrated by plasma membrane disruption which was accom panied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca2+](i) which reached, even afte r 10 min, higher levels in comparison with apoptosis-inducing concentr ations. The loss in membrane integrity observed at these concentration s of TBT could be due to an activation of calcium-dependent phospholip ases. Here it is shown that activation of cytosolic phospholipase A, ( cPLA(2)) leads to liberation of arachidonic acid (AA) out of the phosp holipid membrane. The results presented here demonstrate that organome tals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas hi gh concentrations stimulate necrosis. We suggest that there exists a d irect correlation between the intracellular free calcium concentration and the mode of cell death.