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Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat) - PubMed

  • ️Tue Jan 01 2008

Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat)

Catherine J Walsh et al. Mar Drugs. 2008.

Abstract

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 - 10 microg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 microg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.

Keywords: Karenia brevis; brevetoxin; immunotoxicity; red tide.

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Figures

Figure 1
Figure 1

Percent viability of Jurkat cells treated with PbTx-2, PbTx-3, or PbTx-6 for 24 h. Data are represented as percent of ethanol control viability. Cells treated with PbTx-2 or PbTx-6 had significantly lower viability compared to corresponding ethanol controls and compared with viability of cells treated with PbTx-3 at concentrations indicated by *. Statistical differences compared to ethanol controls were determined using a t-test at each toxin concentration. Comparisons in viabilities among toxin congeners were determined using one-way ANOVA at each concentration. N=4.

Figure 2
Figure 2

Effect of PbTx-2, PbTx-3, and PbTx-6 on cell metabolic activity in Jurkat cells exposed to 0 – 10 μg/ml toxin for 1, 2, or 3 h. Cellular metabolic activity was measured using MTT and reported as percentage of ethanol control. Cells treated with PbTx-2 and -6 had significantly lower metabolic activity compared with corresponding ethanol controls at 5 and 10 μg/ml, as indicated by the *. Cells treated with PbTx-2 or PbTx-6 had significantly lower metabolic activity compared to cells treated with PbTx-3 at 5 and 10 μg/ml, as indicated by the +. *Significantly less (P < 0.05) than ethanol control. +Significantly less (P < 0.05) than treatment with PbTx-3. Changes in cellular metabolic activity were compared to ethanol control for each toxin congener at each concentration using a t-test. Changes in cellular metabolic activity in cells treated with different toxin congeners were determined using one-way ANOVA at each toxin concentration. N=4.

Figure 3
Figure 3

Effect of PbTx-2, PbTx-3, and PbTx-6 on cell metabolic activity in Jurkat cells exposed to 0 – 10 μg/ml toxin for 24 or 48 h. Cellular metabolic activity was measured using MTT and reported as a percentage of ethanol control. Cells treated with brevetoxins had significantly lower metabolic activity compared with corresponding ethanol controls at concentrations indicated by *. Cells treated with PbTx-2 or PbTx-6 had significantly less metabolic activity compared to cells treated with PbTx-3 at concentrations indicated by +. *Significantly less (P < 0.05) than ethanol control. +Significantly less (P < 0.05) than treatment with PbTx-3. Changes in cellular metabolic activity were compared to ethanol control for each toxin congener at each concentration using a t-test. Changes in cellular metabolic activity in cells treated with different toxin congeners were determined using one-way ANOVA at each toxin concentration. N=3.

Figure 4
Figure 4

Caspase-3 activity in Jurkat cells incubated with 0 – 10 μg/ml PbTx-2, PbTx-3, or PbTx-6 for 3 h. St = Staurosporine; EtOH = ethanol control. N =5. Cells treated with PbTx-2 or PbTx-6 had significantly greater (P < 0.05) caspase-3 activity compared with corresponding ethanol controls at the concentrations indicated by *, as determined using a t-test at each concentration. Cells treated with PbTx-2 had significantly greater (P < 0.05) caspase-3 activity compared to cells treated with PbTx-3 at concentrations indicated by +, as determined using a one-way ANOVA at each concentration.

Figure 5
Figure 5

Effect of the caspase-3 inhibitor, Ac-DEVD-CHO, on caspase-3 activity in Jurkat cells exposed to 10 μg/ml PbTx-2 or PbTx-6 for 3 h without inhibitor (No Inh) or with 20 μM of Ac-DEVD-CHO (Inh). The data presented are an average of two trials of the experiment.

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