| The four mammalian cell lines (two normal mammalian cell lines: mouse skin cell line JB6-C41, human hepatocyte cell line HL-7702 and two cancer cell lines: human hepatocellular cancer cell line Bel-7402, mouse neuroblastoma cell line Neuro-2a) were utilized to examine the toxicity of okadaic acid, extraction of Prorocentrum lima, P. donghaiense, Karenia mikimotoi, Alexandrium affine (AC-1 strain) and A. tamarense (AT-6 strain). Their toxic effects on cell proliferation were investigated by MTT assay. And the effects of OA, lipid-soluble component of P. lima and polar-lipid component of K. mikimotoi on morphology and structure of mammalian cells were observed using optical and electron microscope. Furthermore, the induction of OA, P. lima, K. mikimotoi and A. affine on apoptosis or super-oxidation of mammalian cells was detected by Annexin V-FITC & propidium iodide (PI) double-staining, TUNEL assay and TBA assay.The results showed that okadaic acid and the lipid-soluble component of P. lima inhibited the proliferation of JB6-C41, HL-7702, Bel-7402 and Neuro-2a cells significantly in a concentration-dependent manner with 24h IC50 being 100ng/ml, 85ng/ml, 65ng/ml, 70ng/ml and 6000cells/ml, 4000cells/ml, 2000cells/ml, 1000cells/ml, respectively. The polar-lipid component of K. mikimotoi inhibited the proliferation of the four cell lines, while other lipid component and water-soluble fraction had no adverse effects on the proliferation of the four cell lines. The cell-free medium of A. affine (AC-1) inhibited the proliferation of the four cell lines, and the algal cell contents had no adverse effects. The lipid-soluble component of P. donghaiense, the cell-free medium and algal cell contents of A. tamarense (AT-6) all had no adverse effects on the proliferation of the four cell lines. The results indicate that OA and some other toxic substances produced by P. lima, K. mikimotoi or A. affine have adverse effects on the proliferation of mammalian cellsOkadaic acid and lipid-soluble component of P. lima resulted in profound morphological alteration of the four cell lines. The cells rounded up with loss of junctions with neighboring and detached from the substratum. The cell protrusions formed and the cells formed membrane-bounded cell fragments eventually. OA and lipid-soluble component of P. lima also brought about remarkable ultrastructural changes including losing of microvilli, condensation of nuclear chromatin, shedding of cell contents via surface bleb formation, formation of cytoplasmic vacuoles, which all consisted with the characteristics of apoptosis. Detected by Annexin V-FITC & PI double-staining and TUNEL assay, the cells treated with okadaic acid and lipid-soluble component of P. lima all appeared positive fluorescence, which indicates that the four cell lines undergo apoptosis. The results are consonant with the morphological observation. The cell-free medium of A. affine (AC-1) also induced the apoptosis of the four cell lines detected by Annexin V-FITC & PI double-staining. The above results indicate that OA, lipid-soluble component of P. lima and the unknown toxic substance of A. affine (AC-1) might adversely affect the mammalian cells through inducing apoptosis.The polar-lipid component of K. mikimotoi resulted in the cells rounding up, detaching from the substratum, swollen and breaking, which indicates that the cells undergoes necrosis other than apoptosis. The speculation was further proved by Annexin V & PI double-staining. And the malonaldehyde (MDA) contents in the medium of the four cell lines increased significantly. The results indicate that the unknown toxic substance produced by K. mikimotoi could induce the super-oxidation of cells, and its effect mechanism on mammalian cells is different from that of OA, P. lima and A. affine.The sensitivity of four mammalian cell lines to toxins had no obvious differences. Mouse neuroblastma cells (Neuro-2a) and human hepatocellular cancer cells (Bel-7402) were slightly more sensitive. Neuro-2a cell line, usually used in toxicity detection of toxins acting on ion channels such as PSP, was first applied in toxicity detecting of DSP and other toxins. The results indicate that Neuro-2a cells were sensitive to DSP and other toxins and the detecting limit was equivalent to that of other detecting methods including chemical methods and bioassay. Therefore, the cytotoxicity detecting methods using Neuro-2a cells have the potential of being routine monitoring methods for DSP and other toxins due to their sensitivities, easiness to handle, quickness and less need for samples. Neuro-2a cells could be used to detect the toxicities of either PSP or DSP and other toxins, which would make the toxicity evaluation of manifold toxins to be more convenient.In summary, DSP toxins and the main causative species of HABs along the China coast such as K. mikimotoi, Prorocentrum, and Alexandrium might adversely affect the mammalian cells through the induction of apoptosis or super-oxidation by producing toxins. Therefore, the HAB toxins have potential threat to the human health through the food chain transfer, which should be paid more attention. On the other hand, the potential medical value of HAB toxins is deserved to be further investigated for their inhibition effects on mammalian cancer cells.
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