Brevetoxin 2 alters expression of apoptotic, DNA damage, and cytokine genes in Jurkat cells.

Brevetoxins are potent neurotoxins that exert their toxicity through activation of voltage-gated sodium channels. Exposure to brevetoxins cause severe respiratory inflammation in marine mammals and humans. Brevetoxin activation of voltage-gated sodium channels on immune cells can lead to several biological responses including cell proliferation, gene transcription, cytokine production and even apoptosis. Jurkat E6-1 T cells were treated with brevetoxin 2 for 4 hours at a dose previously shown to induce apoptosis and DNA damage. Changes in gene expression were then assessed via PCR arrays. Gene expression analysis revealed significant change in expression of 17 genes related to apoptosis, 21 genes related to DNA damage signaling, and 19 genes encoding common cytokines. The gene expression data supports the idea that brevetoxins trigger complex reactions involving both inflammation and cell death.

Marine brevetoxin induces IgE-independent mast cell activation.

Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H1-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow-derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (ß-hexosaminidase release), intracellular Ca²+ and Na+ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²+ and Na+ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na+ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.

Brevetoxins 2, 3, 6, and 9 show variability in potency and cause significant induction of DNA damage and apoptosis in Jurkat E6-1 cells.

Brevetoxins (PbTx) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis, an organism linked to periodic red tide blooms. Brevetoxins exert their toxicity by interacting with neurotoxin receptor site five associated with domain IV of the alpha subunit of the voltage gated sodium channel. Brevetoxin binding to tissues that contain voltage gated sodium channels on excitable cells results in membrane depolarization, repetitive firing, and increase in sodium currents. Brevetoxins have been linked to deaths in marine mammals, which are exposed through ingestion of organisms harboring high brevetoxin concentrations and through the inhalation of aerosolized brevetoxins. Humans are also at risk, primarily through respiratory exposure which can result in a severe inflammatory response. The purpose of this study was to determine the effect of four brevetoxins on Jurkat E6-1 cell proliferation, to assess their variability in potency, genotoxicity, and to determine if brevetoxin causes cell death, specifically through an apoptotic or necrotic mechanism. PbTx 2, 3, 6, and 9 were tested at concentrations of 10(-4)-10(-12) M to determine the IC(50) values and effect on cell proliferation. The IC(50) concentration was then used in the single cell gel electrophoresis assay to determine genotoxicity. The ability to induce apoptosis was then assessed with the Vybrant apoptosis assay, caspase activation assays and PARP cleavage. Results from the cellular proliferation assays demonstrated that high doses of PbTxs inhibit the ability of Jurkat cells to proliferate while lower doses caused an increase in proliferation and that PbTx2 is the most cytotoxic brevetoxin followed by brevetoxins 6, 3, and 9. Brevetoxins 2, 3, and 6 all caused significant DNA damage. A 4 h exposure to brevetoxins 2, 3, 6, and 9 at values close to the IC(50) values resulted in apoptosis positive staining in Jurkat E6-1 cells. High doses of brevetoxins 2 and 6 resulted in activation of caspases 3/7 and 8 and cleavage of poly (ADP-ribose) polymerase (PARP). The conclusions are that brevetoxins affect cell proliferation in a dose-dependent fashion, are genotoxic, and cause cell death through an apoptotic mechanism.

Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood.

BACKGROUND: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability. METHODS: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables. RESULTS: Storage of blood samples for >1 week at 4 degrees C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex. CONCLUSION: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

Effect of conazole fungicides on reproductive development in the female rat.

Three triazole fungicides were evaluated for effects on female rat reproductive development. Rats were exposed via feed to propiconazole (P) (100, 500, or 2500 ppm), myclobutanil (M) (100, 500, or 2000 ppm), or triadimefon (T) (100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 98. Body weight (BW) and anogenital distance (AGD) at PND 0, age and BW at vaginal opening (VO), estrous cyclicity, and body and organ weight at necropsy were measured. BW at PND 0 was unaffected by treatment. AGD was increased by M2000. VO was delayed by M2000 and T1800. Estrous cyclicity was initially disrupted by P500, P2500 and T1800, but later normalized. At PND 99 there was a decrease in BW by T1800, an increase in liver weight by P2500 and T1800, and an increase in ovarian weight by M2000 and T1800. It is concluded that exposure to P, M and T adversely impacted female rodent reproductive development.

Gene expression in head hair follicles plucked from men and women.

Characterizing gene expression in hair follicles can help to elucidate the hair growth cycle by delineating the genes and pathways involved in follicular growth and degeneration. The objectives of this study were to determine whether intact RNA could be extracted from a small number of plucked, unstaged hair follicles in sufficient quantity to conduct gene expression profiling, and to conduct global gene expression profiling. To this end, RNA was extracted from 1 to 3 unstaged follicles plucked from the scalp of 36 volunteers. The average quantifiable yield of RNA/follicle was 112.5 ng. Ribosomal ratios were lower than normally expected, but investigation indicated the RNA was intact. Ten of the samples were amplified and hybridized to Affymetrix genechips. On average, 2,567 of the total probe sets (8,500) were expressed in each sample; 1,422 were expressed in all 10 samples; 97 were significantly changed in one gender compared to the other, and 41 had high levels of interindividual variability. This study demonstrates that RNA of sufficient quantity and quality to use in microarray hybridizations can be obtained from as little as a single plucked human hair follicle. Genes expressed in all individuals are probably related to follicular growth and could form a starting set for developing signatures of toxicant exposure. The differentially expressed genes could be involved in producing gender and interindividual differences in hair growth.