Abstract

Effects of Brevetoxin-3 (PbTx-3) on excitatory tissues have been extensively studied, however, its cellular effects on epithelial linings following environmental exposure remain largely unknown. The respiratory epithelium of man and marine mammals is exposed to varying concentrations of aerosolized PbTx-3 during Karenia brevis blooms. Epithelial damage predisposes individuals to infectious secondary pathogenic invasion and thus additional morbidity and mortality. To investigate if PbTx-3 induces DNA damage and oxidative DNA lesions, in vitro studies were conducted using a 'normal' human bronchial epithelial cell line (BEAS-2B) as a model for marine mammal effects. Because symptoms of brevetoxin exposure are often worse in individuals with respiratory compromise, a neoplastic human respiratory epithelial cell line (H460) was also used as a model for respiratory compromise. A comet assay was used to determine if DNA damage occurs following exposure to 10, 100 and 500 ng/ml PbTx-3 for 1 hour at 37°C. Post-exposure, some cell loss was apparent at higher PbTx-3 concentrations. For BEAS-2B cells, the mean negative control tail moment was 6.05 (SE = ±1.14), the hydrogen peroxide-treated (H2O2) positive control was 53.98 (3.34), PbTx-3 at 10 ng/ml was 8.89 (0.82), at 100 ng/ml was 12.62 (1.42), and at 500 ng/ml was 6.81 (0.93). For H460 cells, the mean negative control tail moment was 0.30 (0.08), the positive control was 62.56 (14.14), PbTx-3 at 10 ng/ml was 11.16 (1.06), at 100 ng/ml was 0.13 (0.02), and at 500 ng/ml was 0.21 (0.05). The data indicates that exposure to PbTx-3 results in acute DNA damage in respiratory epithelial cells and may result in acute cell loss at higher concentrations. It also suggests that significant genotoxic damage can occur at lower PbTx-3 concentrations (10 ng/ml) in neoplastic respiratory epithelial cells, whereas higher concentrations (100 ng/ml) may be required to induce significant damage in uncompromised respiratory epithelial cells. A formamidopyrimidine-DNA glycosylase (Fpg) fragment length analysis using repair enzymes (FLARE) assay was used to determine if PbTx-3 induces oxidative DNA lesions following exposure for 20 minutes at 4°C. For Fpg-untreated BEAS-2B cells, the mean negative control tail moment was 4.37 (0.77), the H2O2-treated positive control was 8.55 (0.68), PbTx-3 at 10 ng/ml was 7.49 (0.70), at 100 ng/ml was 9.95 (0.74), and at 500 ng/ml was 9.32 (0.62). For Fpg-treated BEAS-2B cells, the mean negative control tail moment was 7.73 (0.80), the positive control was 9.79 (0.70), PbTx-3 at 10 ng/ml was 7.20 (0.73), at 100 ng/ml was 9.27 (0.76), and at 500 ng/ml was 9.17 (0.64). For Fpg-untreated H460 cells, the mean negative control tail moment was 6.48 (0.85), the positive control was 16.99 (0.64), PbTx-3 at 10 ng/ml was 2.69 (0.67), at 100 ng/ml was 4.08 (0.65), and at 500 ng/ml was 4.27 (0.67). For Fpg-treated H460 cells, the mean negative control tail moment was 3.45 (0.97), the positive control was 20.10 (0.73), PbTx-3 at 10 ng/ml was 3.41 (0.78), at 100 ng/ml was 3.20 (0.75), and at 500 ng/ml was 4.40 (0.77). FLARE data indicates that PbTx-3 exposure results in acute DNA damage in respiratory epithelial cells, and that significant oxidative lesions are induced at higher PbTx-3 concentrations (500 ng/ml) in neoplastic respiratory epithelial cells.