Effects of Brevetoxins on Murine Myeloma SP2/O Cells: Aberrant Cellular Division

Abstract

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

Effects of in vitro Brevetoxin Exposure on Apoptosis and Cellular Metabolism in a Leukemic T Cell Line (Jurkat)

“…Depending on toxin congener used, concentrations as low as 2.5 μg/ml significantly decreased this response compared to control. Han et al [ 23 ] reported a transient increase in cellular activity after 1.5 h exposure to brevetoxin, an effect which was not observed in the present study. Han et al used crude toxin extract at low concentrations, which may account for the observed differences.…”

“…Several potential mechanisms of action on immune system components have been suggested, including inhibition of cathepsin active sites [ 36 , 37 ], induction of apoptosis [ 16 , 22 ], and release of inflammatory mediators [ 16 ]. Effects on the cell cycle have also been hypothesized, based on reports of reduced lymphocyte proliferation in manatees [ 17 ], aberrant cell division in a cell line (SP2/0) exposed to brevetoxin in vitro [ 23 ], and inhibition of cellular proliferation of CHO-K1-BH4 cells [ 24 ]. In this paper, we address effects on brevetoxin on cellular metabolic activity and apoptosis.…”

“…Previously published reports on effects of brevetoxin on cell proliferation have included use of a reagent based on mitochondrial dehydrogenase activity, WST-1 [ 23 ], and determination of cell number [ 24 ]. MTT, a measure of metabolic activity, specifically mitochondrial function, of viable cells, has been used as a measure of cell proliferation [ 38 – 40 ] in other systems.…”

“…Demonstrated immune functional impairment resulting from brevetoxin exposure includes reduced phagocytosis [ 19 ], decreased plaque-forming ability [ 19 , 20 ], and decreased lymphocyte proliferation [ 17 ]. In vitro brevetoxin exposure has been reported to result in DNA damage in human lymphocytes [ 22 ] and to affect the growth of cell lines [ 23 , 24 ].…”

Effects of in vitro Brevetoxin Exposure on Apoptosis and Cellular Metabolism in a Leukemic T Cell Line (Jurkat)

“…Effects on the cell cycle have also been hypothesized, based on reports of reduced lymphocyte proliferation in manatees [17], aberrant cell division in a cell line (SP2/0) exposed to brevetoxin in vitro [23], and inhibition of cellular proliferation of CHO-K1-BH4 cells [24]. In this paper, we address effects on brevetoxin on cellular metabolic activity and apoptosis.…”