Comparative Biochemistry and Physiology Part C 124 (1999) 221 – 226 Characterization of hemolytic activity of 3-alkylpyridinium polymers from the marine sponge Reniera sarai Petra Malovrh, Kristina Sepc ˇic ´ *, Tom Turk, Peter Mac ˇek Department of Biology, Biotechnical Faculty, Uniersity of Ljubljana, Vec ˇna pot 111, 1111, Ljubljana, Sloenia Received 16 February 1999; received in revised form 28 July 1999; accepted 3 August 1999 Abstract Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as potent anticholinesterase agents; in addition they show moderate hemolytic and cytotoxic activities. The hemolytic activity of poly-APS is due to their detergent-like structure and behavior in aqueous solutions. In this work, the hemolytic activity of poly-APS is analyzed and compared to that of structurally-related monomeric cationic surfactants. The influence of different divalent cations and lipids on poly-APS induced hemolysis is discussed. The dimensions of lesions caused by poly-APS in erythrocyte membranes are determined by the use of osmotic protectants. Finally, the possible role of poly-APS in their natural environment is proposed. © 1999 Elsevier Science Inc. All rights reserved. Keywords: 3-Alkylpyridinium polymers; Cationic surfactant; Detergent; Hemolytic activity; Marine sponge; Reniera sarai www.elsevier.com/locate/cbpc 1. Introduction Marine sponges produce a variety of antiviral, an- tibacterial, cytotoxic, cytolytic, and hemolytic com- pounds. As hemolytic were proved proteins [8,9,17], saponin-like peptides [23], and non-identified com- pounds [7,19]. Cytolytic agents, water soluble polymeric 3-octylpyridinium salts (poly-APS) (see Fig. 1), were isolated from the marine sponge Reniera sarai [26,27]. Poly-APS are structurally and functionally related to halitoxins, 0.5 – 25 kDa 3-alkylpyridinium polymers iso- lated from several sponges of the genus Haliclona [5,24]. Poly-APS were determined to be a mixture of 29 (5520 Da) and 99 (18 900 Da) polymerized 3-octylpyri- dinium units [27]. In halitoxins, pyridinium rings are connected by methyl-branched alkyl chains composed of 8–11 carbon atoms instead of the octyl chain in poly-APS. To date, 3-alkylpyridinium oligomers and polymers have been reported to be cytotoxic [13,24,26,31], hemolytic [5,24,26], antibacterial [24], an- tifeeding [1], toxic to animals [5,24,31], antimitotic [5,13], neurotoxic [5], and inhibitory for acetyl- cholinesterase [27 – 29], epidermal growth factors [10] and muscarinic receptors [12]. In aqueous solution, poly-APS form larger structures with an average hydrodynamic radius of 23 3 nm (mean SEM) and surfactant-like characteristics [27]. Our preliminary study has shown that besides a potent and complex anticholinesterase activity [28], poly-APS are hemolytic and cytotoxic for different cell lines in- cluding the transformed ones [26]. This study addresses the characteristics of hemolytic activity of poly-APS as compared to hemolysis induced by the chemically re- lated pyridinium surfactants, cetylpyridinium chloride (CPC), and cetyltrimethylammonium bromide (CTAB). Cationic surfactants are known as hemolytic com- pounds [15,32,34] and are also used as antibacterial or plaque-inhibiting agents [2]. We suggest that 3- alkylpyridinium polymers, displaying a broad spectrum of biological activities, may serve as unspecific defense agents in marine sponges. Abbreiations: CMC, critical micelle concentration; CPC, cetylpyri- dinium chloride; CTAB, cetyltrimethylammonium bromide; HC 0.5 , concentration of surfactant causing 50% hemolysis in 2 min; IC 0.5 , cation concentration producing 50% inhibition of hemolysis; PEG, polyethyleneglycol; Poly-APS, polymeric alkylpyridinium salts. * Corresponding author. Tel.: +386-61-1233388; fax: +386-61- 273390. E-mail address: kristina.sepcic@uni-lj.si (K. Sepc ˇic ´) 0742-8413/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII:S0742-8413(99)00067-5