Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur)

Abstract

Two species of venomous pelagic cnidaria are compared according to their enzymatic, cytotoxic and haemolytic potency. The widely distributed jellyfish Cyanea capillata and Cyanea lamarckii were collected in the North Sea at the coasts of the Orkney Island and the Island of Helgoland. Purified cnidocyst extracts from fishing and mesenteric tentacles were prepared and tested for their bioactivity.

The haemolysis induced by toxins of C. capillata was determined with respect to organism size and toxigenic organs. The haemolytic activity of the related species C. lamarckii was documented for the first time. Dose dependent haemolytic activities have been detected by means of protein equivalents at concentrations above 20 μg_protein/mL. Extracts of fishing tentacle cnidocysts showed a less potent haemolytic activity compared to extracts of mesenteric tentacles. In vitro studies with permanent cells of a hepatoma cell line have shown a time and concentration dependent loss of cell vitality up to 90% at 33.3 μg_protein/mL (10 μg_protein/10⁵ cells). Supplementing the cell based toxicity tests an enzyme assay was performed to measure a phospholipase A₂ (PLA₂) activity. A PLA₂-like activity could be demonstrated in cnidocysts extracts prepared from mesenteric and fishing tentacles of both jellyfish species.

Introduction

Species of the phylum cnidaria are major components of the pelagic system. The jellyfish Cyanea capillata and Cyanea lamarckii belong to the most abundant scyphocoan jellyfish in the northern coastal zone. Scyphomedusae are efficient predators. They express successful foraging behaviour and a wide dietary range. Studies on the trophodynamics have shown an enormous predation on fish eggs, fish larvae and small fish. Another important interaction is the potential competition on prey among cnidaria and zooplanktonivorous fish species especially when they occur as jellyfish blooms (Bamstedt et al., 1994; Purcell and Arai, 2001).

A further concern is the stinging capacity of jellyfish and the resulting public health hazard. At tropical and subtropical coast lines life-threatening and frequent contact between humans and jellyfish are an ongoing field of attention (Bailey et al., 2003).

Envenomations in the northern coastal territories are considered to be much less serious, although with the observed larger population numbers an increasing amount of stings is expected (Mills, 2001; Burnett, 2001). The cnidarian venom is preserved in specialized cell organelles (cnidocysts) which are harboured in cnidocytes situated as batteries at fishing and mesenteric tentacles. Adequate chemical and mechanical signals induce the fast discharge of cnidocysts resulting in immediate paralysis of its prey. Human can respond with cutaneous irritations and pain up to cardiovascular system failure after accidental contact with Cyanea spec. (Burnett, 2001). The reddish-brown pigmented C. capillata is more common and more information on its economic relevance and toxic potency is available (Burnett and Calton, 1987; Heeger et al., 1992). A few in vitro and in vivo studies with crude and partially purified C. capillata venom demonstrate cytolytic and cardiotoxic potencies (Walker, 1977a, Walker, 1977b; Walker et al., 1977; Long and Burnett, 1989). In these investigations mainly cnidocysts of tentacles were used and no special attention was paid on the effect of oral arms or of the surrounding tissue. The venom of C. capillata consists of a broad range of proteinaceous substances and the cardiotoxicity seems to be associated to a 70.000 Da protein whereas the hemolytic activity is supposed to be related to peptides (Walker, 1977a, Walker, 1977b; Long and Burnett, 1989), but causative substances could not finally be isolated and characterized. Comparable in vitro or in vivo studies for the toxicity of C. lamarckii are not performed, although the stinging capacity is specified as low.

The complex nature of the venom and the supposed multitude of pharmacological effects require the application of various in vitro assays to cover different modes of effects. The present paper is a comparative study comprising of two scyphozoan jellyfish species of different sizes, different types of toxigenic organs with a haemolysis assay detecting membrane activity and a cell line-based assay to measure cytotoxicity.

The haemolytic action of venoms is often accompanied by an enzymatic phospholipase A₂ activity and a result of synergistic protein action (Hessinger and Lenhoff, 1976). Phospholipase A₂ hydrolyzes phospholipids as components of cell membranes at the sn-2 position leading to the cleavage of lysophospholipids and free fatty acids. Metabolites of the fatty acid residue arachidonic acid are well known as secondary messengers in signal transduction and inflammation and control a wide variety of cell functions (Six and Dennis, 2000).

A number of marine invertebrates among them cnidaria have been screened for PLA₂ activity. But there is inconsistent information on a PLA₂ activity in C. capillata available and no information for C. lamarckii. Whereas Nevalainen et al. (2004a) described a relatively high activity in whole tissue extracts of C. capillata compared to other cnidaria Vaskovski and Suppes (1972) did not detect any activity with a less sensitive Thin Layer Chromatography method. Cnidarian PLA₂ may be involved in physiological cell membrane lipid metabolism, in the irritation at the stinging site and in the enhancement of the haemolytic activity.