Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 1. Effects of controls in cell line panel, red blood cells, and the enzyme protein phosphatase 2A. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 800 nM brevetoxin B, 1.5 µM cylindrospermopsin, 500 nM domoic acid, 100 nM microcystin LR, and 10 nM okadaic acid for 72 h. The results were normalized to solvent controls and expressed as a per- centage. Those that induce more than 50% cytotoxicity (dotted line) are considered active. In addition, sheep red blood cells were treated with the same compounds for 4 h to measure hemagglutination or 6 h to measure cell lysis. None of the controls exhibited red blood cell lysis or hemagglutination at the concentrations tested. The effect of these compounds on protein phosphatase after 30 min of treatment is also shown. Unique patterns of cytotoxicity were seen with each of the known phycotoxins tested. The average of three independent experiments ± standard deviation is shown.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Solvent, Lysis, Red Blood Cell Lysis, Standard Deviation

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 2. Effects of southernmost samples (sites 1–5) in cell line panel, red blood cells, and the enzyme protein phosphatase 2A. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effects of 10 µg/mL samples on protein phosphatase after 30 min of treatment are also shown. Those that induce more than 50% cytotoxicity (dotted line) are considered active. These samples exhibited cytotoxicity throughout all the sampling periods. During year 1, a Microcystis bloom occurred, and the pattern of cytotoxicity for microcystin LR is seen in sites 1–4. During years 2 and 3, cytotoxicity was observed but did not match any patterns, suggesting it was caused by either a mixture of toxins or an emerging toxin. The average of three samples ± standard error of the mean is shown. An asterisk denotes statistically significant differences (p ≤0.05) to fall year 1 data through Student’s t-test.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Lysis, Sampling

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 3. Effects of southern middle samples (sites 6–10) in cell line panel, red blood cells, and the enzyme protein phosphatase 2A. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effect of 10 µg/mL samples on protein phosphatase after 30 min of treatment is also shown. Those that induce more than 50% cytotoxicity (dotted line) are considered active. During year 1, site 6 (Jensen Beach) exhibited strong cytotoxicity. This was transient, as it was not seen at later times. Cytotoxicity observed in these sites did not match any patterns, suggesting it was caused by either a mixture of toxins or an emerging toxin. The average of three samples ± standard error of the mean is shown. An asterisk denotes statistically significant differences (p ≤0.05) to fall year 1 data through a Student’s t-test.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Lysis

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 4. Effects of northern middle samples (sites 11–15) in cell line panel, red blood cells, and the enzyme protein phosphatase 2A. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effect of 10 µg/mL samples on protein phosphatase after 30 min of treatment is also shown. Those that induce more than 50% cytotoxicity (dotted line) are considered active. Overall, these appear to be healthier sites as less cytotoxicity was seen in these sites, except for sites 14 and 15, which exhibited some cytotoxicity in years 2 and 3. Cytotoxicity observed in these sites appears to match the pattern seen when testing samples from a Pyrodinium bloom. The average of three samples ± standard error of the mean is shown. An asterisk denotes statistically significant differences (p ≤0.05) to Fall year 1 data through a Student’s t-test.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Northern Blot, Lysis

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 5. Effects of northern samples (sites 16–20) in cell line panel, red blood cells, and the enzyme protein phosphatase 2A. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effects of 10 µg/mL samples on protein phosphatase after 30 min of treatment are also shown. Those that induced more than 50% cytotoxicity (dotted line) are considered active. Overall, these appear to be healthier sites as less cytotoxicity was seen in these sites, except for sites 17 and 19, which exhibited some cytotoxicity in years 2 and 3. Cytotoxicity observed in these sites did not match any patterns, suggesting it was caused by either a mixture of toxins or an emerging toxin. The average of three samples ± standard error of the mean is shown. An asterisk denotes statistically significant differences (p ≤0.05) to fall year 1 data through a Student’s t-test.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Northern Blot, Lysis

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 6. (a) Cytotoxicity patterns of samples collected during a Pyrodinium bloom. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effects of 10 µg/mL samples on protein phosphatase after 30 min of treatment are also shown. Those that induced more than 50% cytotoxicity (dotted line) are considered active. A consistent pattern of cytotoxicity was seen in these samples. The average of three samples ± standard deviation is shown. (b) Comparison of LC-HRMS chromatograms of cytotoxic bloom and non-cytotoxic, non-bloom samples from the NIRL sites. The compounds at m/z 800.6011 and 700.5698 are only found in cytotoxic bloom samples and were originally proposed to be responsible for the observed cytotoxicity. Further separation by preparative HPLC led to the observation that the cytotoxic compound is a very minor component of the extract. The compounds with m/z 800.6011 and 700.5698 are present in all bloom samples from these sites and may have other roles as allelochemicals.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Lysis, Standard Deviation, Comparison

Journal: Toxins

Article Title: An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

doi: 10.3390/toxins15110664

Figure Lengend Snippet: Figure 7. (a) Cytotoxicity patterns of a sample collected during a Microcystis bloom in Lake Okee- chobee. OATP1 A2 and OATP1 B1 Transporto™cells and Vero, Hep G2, and SH-SY5Y cells were treated with 10 µg/mL samples for 72 h. Sheep red blood cells were treated with 50 µg/mL samples for 4 h to measure hemagglutination or 6 h to measure cell lysis. The effects of 10 µg/mL samples on protein phosphatase after 30 min of treatment are also shown. Those that induced more than 50% cy- totoxicity (dotted line) are considered active. Sample 41971 is the crude extract, and the other samples shown are fractions derived from the crude extract, with 41972 and 41974 containing microcystins. Sample 41974 is highly enriched in microcystin LR and exhibits the same pattern of cytotoxicity as authentic microcystin LR control. The average of two replicates ± standard deviation is shown. (b) Comparison of LC-HRMS chromatograms for samples at 4 sites in the St Lucie estuary and SIRL during the 2018 Microcystis bloom and from Lake Okeechobee 2021. Blue arrows designate the peak for microcystin LR (MLR). The lake and site 2 had the highest concentrations of microcystin LR.

Article Snippet: Treatments consisted of 50 μg/mL water samples, solvent controls, phycotoxin controls, 25 μg/mL anti-sheep red blood cell antibody (assay control, 213–4139, Rockland Immunochemicals, Pottstown, PA, USA), and 50 μg/mL concanavalin A (positive control; C0412, Sigma, St. Louis MO, USA).

Techniques: Lysis, Derivative Assay, Control, Standard Deviation, Comparison