Hydrogen Peroxide Induces Apoptotic-Like Cell Death in Coelomocytes of Themiste petricola (Sipuncula) GUILLERMO A. BLANCO 1, * ,¶ , JUANITA BUSTAMANTE 2,¶ , MARIANA GARCIA 1,# AND SILVIA E. HAJOS 1,¶ 1 Department of Immunology, IDEHU-National Research Council (CONICET); and 2 Department of Physicochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), Buenos Aires, Argentina Abstract. Apoptosis is an active form of cell death that plays a critical role in physiological and pathological con- ditions of multicellular organisms. These conditions include development, organogenesis, and elimination of infected, mutated, or damaged cells. Sipunculan cells may respond to changes in environmental exposure to oxidative stress by induction of apoptotic cell death. In coelomocytes of the sipunculan worm Themiste petricola, we evaluated morpho- logical and biochemical changes that were induced by hy- drogen peroxide (H 2 O 2 ) and that could be compatible with an apoptotic-like phenotype. At an exposure of 100 mM H 2 O 2 , coelomocytes exhibited several morphological hall- marks of apoptosis such as chromatin condensation, nuclear segmentation, cell volume decrease, membrane blebbing, and formation of apoptotic bodies. Biochemical evidences of apoptotic-like cell death included exposure of phospha- tidylserine (PS) in the outer leaflet of the plasma membrane and oligonucleosomal DNA fragmentation. In addition, ex- posure of coelomocytes to H 2 O 2 induced a rapid massive loss of mitochondrial membrane potential and of the acidic pH of lysosomes. Overall, our results showed that, in sipun- culan coelomocytes, H 2 O 2 can induce changes compatible with an apoptotic-like phenotype. The finding of an oxida- tive-stress-induced apoptotic-like phenotype in a sipunculan worm may indicate that this kind of cell death process participates in regulation of cell number during physiolog- ical and pathological situations, including immune re- sponses. Introduction Apoptosis is an active form of cell death with a vital role in physiological and pathological conditions throughout the development and adult life of multicellular organisms, re- moving infected, injured, or mutated cells (Kerr et al., 1972; Wyllie et al., 1980; Ellis and Horvitz, 1986). It is charac- terized by a series of morphological events such as cell shrinkage, chromatin condensation, nuclear and cytoplas- mic fragmentation, and formation of dense bodies that are rapidly phagocytosed by neighboring cells (Vaux and Strasser, 1996). These typical morphological changes result from the activation of biochemical events such as increased intracellular Ca ++ concentrations, uncoupling of mitochon- drial electrical potential, fragmentation of oligonucleosomal DNA, and proteolytic degradation of specific substrates (Wyllie, 1980; Schwartzman and Cidlowski, 1993; Zheng et al., 1998; Enari et al., 1998). It is generally assumed that apoptosis appeared early in the evolution of multicellular animals, and several studies have shown that the compo- nents of the cell death pathway are highly conserved throughout the evolution of Caenorhabditis elegans, Dro- sophila, and mammals (Meier et al., 2000; Vernooy et al., 2000). Apoptosis can be induced under several stress conditions such as withdrawal of growth factor (Xia et al., 1995), exposure to cytotoxic drugs (Muschel et al., 1998; Lopes et al., 2003), or loss of normal adhesion to extracellular matrix (Frisch and Francis, 1994). Environmental pollutants such as heavy metals and organotines are known to induce apo- Received 11 July 2005; accepted 26 October 2005. * To whom correspondence should be addressed. E-mail: gblanco@ ffyb.uba.ar ¶ Members of the National Research Career (CONICET). # Fellow from UBA. Abbreviations: AO, acridine orange; EB, ethidium bromide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide;  m , mitochondrial transmembrane potential; PI, propidium iodide; PS, phosphatidylserine. Reference: Biol. Bull. 209: 168 –183. (December 2005) © 2005 Marine Biological Laboratory 168