Linkage, Empowerment, and Access to Prevent Hypertension: A Novel Program to Prevent Hypertension and Reduce Cardiovascular Health Disparities in Detroit, Michigan.

BACKGROUND: Serious cardiovascular health disparities persist across the United States, disproportionately affecting Black communities. Mounting evidence supports negative social determinants of health (SDoH) as contributing factors to a higher prevalence of hypertension along with lower control rates. Here, we describe a first-of-a-kind approach to reducing health disparities by focusing on preventing hypertension in Black adults with elevated blood pressure (BP) living in socially vulnerable communities. METHODS AND RESULTS: Linkage, Empowerment, and Access to Prevent Hypertension (LEAP-HTN) is part of the RESTORE (Addressing Social Determinants to Prevent Hypertension) health equity research network. The trial will test if a novel intervention reduces systolic BP (primary outcome) and prevents the onset of hypertension over 1 year versus usual care in 500 Black adults with elevated BP (systolic BP 120-129 mm Hg; diastolic BP <80 mm Hg) in Detroit, Michigan. LEAP-HTN leverages our groundbreaking platform using geospatial health and social vulnerability data to direct the deployment of mobile health units (MHUs) to communities of greatest need. All patients are referred to primary care providers. Trial participants in the active limb will receive additional collaborative care delivered remotely by community health workers using an innovative strategy termed pragmatic, personalized, adaptable approaches to lifestyle, and life circumstances (PAL2) which mitigates the impact of negative SDoH. CONCLUSIONS: LEAP-HTN aims to prevent hypertension by improving access and linkage to care while mitigating negative SDoH. This novel approach could represent a sustainable and scalable strategy to overcoming health disparities in socially vulnerable communities across the United States.

Does Vitamin D Provide Added Benefit to Antihypertensive Therapy in Reducing Left Ventricular Hypertrophy Determined by Cardiac Magnetic Resonance?

BACKGROUND: Left ventricular hypertrophy (LVH) and vitamin D deficiency have been linked to hypertension (HTN) and cardiovascular disease, particularly in African Americans (AAs). Our objective was to determine if the addition of vitamin D to antihypertensive therapy would lead to greater regression of LV mass index (LVMI) as determined by cardiac magnetic resonance (CMR) after 1 year in vitamin D deficient AA patients with uncontrolled HTN and LVH. METHODS: This study was a randomized, double-blind, placebo-controlled, single-center study. AA patients with HTN (systolic blood pressure [BP] >160 mm Hg), increased LVMI, and vitamin D deficiency (<20 ng/ml) were randomized. All patients received antihypertensive therapy combined with biweekly 50,000 IU vitamin D3 (vitamin D group, n = 55) or placebo (placebo group, n = 58). RESULTS: At 1 year, there were no statistical differences between the vitamin D and placebo groups in LVMI (-14.1 ± 14.6 vs. -16.9 ± 13.1 g/m2; P = 0.34) or systolic BP (-25.6 ± 32.1 vs. -25.7 ± 25.6 mm Hg; P = 0.99) reduction, respectively. Serum vitamin D levels increased significantly in the vitamin D group compared with placebo (12.7 ± 2.0 vs. 1.8 ± 8.2 ng/ml; P < 0.001). CONCLUSIONS: In this high-risk cohort of AAs we did not find an association between vitamin D supplementation and differential regression of LVMI or reduction in systolic BP. However, our study suffered from a small sample size with low statistical power precluding a definitive conclusion on the therapeutic benefit of vitamin D in such patients. CLINICAL TRIALS REGISTRATION: Trial Number NCT01360476. Full trial protocol is available from corresponding author.

Nitroglycerin Use in the Emergency Department: Current Perspectives.

Nitroglycerin, a fast-acting vasodilator, is commonly used as a first-line agent for angina in the emergency department and to manage chest pain due to acute coronary syndromes. It is also a treatment option for other disease states such as acute heart failure, pulmonary edema, and aortic dissection. Nitroglycerin is converted to nitric oxide, a potent vasodilator, in the body, leading to venodilation at lower dosages and arteriodilation at higher dosages that results in both preload and afterload reduction, respectively. Although nitroglycerin has historically been administered as a sublingual tablet and/or spray, it is often given intravenously in the emergency department as this enables titration to effect with predictable pharmacokinetics. In this review article, we outline the indications, mechanism of action, contraindications, and adverse effects of nitroglycerin as well as review relevant literature and make general recommendations regarding the use of nitroglycerin in the emergency department.

Public Housing Resident Perspectives on Smoking, Barriers for Smoking Cessation, and Changes in Smoking Mandates.

BACKGROUND: Individuals from low-income groups report disproportionate rates of cigarette use, secondhand smoke (SHS) exposure with increased morbidity and mortality. Smoking bans in public housing have been enacted in attempt to reduce tobacco use and SHS exposure among lower income individuals. This study investigated the support needs of tobacco users living in two public housing complexes in Detroit, Michigan (USA), including their perspectives on smoking, resources and barriers for smoking cessation, and the impact of policy changes. METHODS: This is a mixed-methods study, using a qualitative focus groups approach and a short survey, public housing residents interview data was analyzed to explore themes related to smoking-related issues. Specifically, six themes were assessed across four focus groups: (1) Quitting Smoking, (2) Current Smoking Cessation Resources, (3) Legal Mandates, (4) Education and Perceptions of Smoking, (5) Community Needs and Barriers, and (6) Medical Experiences. RESULTS: There were 59 participants; the majority (39/42, 93%) of smokers reported at least one quit attempt. During the focus groups, several participants indicated a desire to quit smoking but reported barriers to smoking cessation, such as lack of access to medications, social triggers to continue smoking, and socioeconomic stressors. A number of suggestions were provided to improve smoking cessation resources, including support groups, graphic images of smoking-related diseased tissue, and better communication with health care providers. CONCLUSIONS: These findings demonstrate smoking bans in two public housing complexes can be effective yet are dependent upon a complex set of issues, including numerous barriers to care.

Randomized Controlled Trial of a Novel Silicone Device for the Packing of Cutaneous Abscesses in the Emergency Department: A Pilot Study.

OBJECTIVE: Traditional treatment of abscesses in the emergency department includes packing with gauze; however, this can add pain and discomfort to the procedure and frequently involves a follow-up visit for packing removal. Alternatives to gauze packing have been proposed, but they may have disadvantages in the context of emergency care. The objective of this pilot study was to investigate the use of a novel silicone packing device - the Derma-Stent. METHODS: This was a randomized controlled pilot study of 50 patients from two urban emergency departments with uncomplicated superficial abscesses. The primary outcome was the likelihood of self-removal of packing gauze versus the silicone device. Secondary outcome measures included subjective clinician and patient metrics, such as pain and ease of use. RESULTS: Patients identified with simple cutaneous abscesses were randomized to intervention (packing with the novel silicone device, n=25) or standard care (gauze packing, n=25). Mean age was 36 years, 54% were female, and 96% identified as African American. Although it took longer to place, the silicone device (19.0 vs 15.3 minutes, p=0.03), pain scores were significantly lower (4.3/10 vs 7.1/10, p=0.008) and ease of use reported by physicians better in the silicone-device group (4.8/5 vs 4.0/5, p=0.002). A high unknown rate in the gauze-packing group limits discussion of the likelihood of self-removal; however, the silicone device was more likely to remain in place (60%) versus the gauze packing at 3 days (24%, p=0.01). CONCLUSION: This pilot randomized controlled trial compared the treatment of packing cutaneous abscesses with gauze versus using the silicone device. Limitations in the data prevent discussion on likelihood of self-removal. However, the silicone device was more likely to remain in place at day 3 follow-up and was equally effective to gauze packing in abscess reduction while also improving patient-reported pain scores. It did take longer to place the silicone device; however, physicians reported better ease of use and removal. This pilot study is encouraging for additional larger-scale trials that are required to further assess the utility of this device in the emergency department.

Ultra-high dose intravenous nitroglycerin in an ESRD patient with acutely decompensated heart failure.

Acute cardiogenic pulmonary edema is a highly unstable and potentially lethal condition that is most commonly associated with markedly elevated blood pressure (BP). Use of nitrates, diuretics, and non-invasive positive pressure ventilatory support are the mainstays of early intervention and stabilization. Use of high-dose bolus intravenous nitroglycerin, which causes both preload and afterload reduction, has shown significant promise in studies to date, reducing the need for endotracheal intubation (ETI) and intensive care unit admission. To date, the highest recorded total dose of nitroglycerin used during the initial stabilization of acute pulmonary edema has been 20 mg. Here, we describe a patient with end-stage renal disease who developed acute cardiogenic pulmonary edema and received a total of 59 mg nitroglycerin (56 mg push dose intravenous + 3 mg intravenous drip) over 41 minutes leading to successful stabilization and avoidance of ETI, facilitating rapid initiation of emergent hemodialysis.

Angiotensin-converting enzyme inhibitors increase anti-fibrotic biomarkers in African Americans with left ventricular hypertrophy.

Angiotensin-converting enzyme inhibitors (ACEi) are part of the indicated treatment in hypertensive African Americans. ACEi have blood pressure-independent effects that may make them preferred for certain patients. We aimed to evaluate the impact of ACEi on anti-fibrotic biomarkers in African American hypertensive patients with left ventricular hypertrophy (LVH). We conducted a post hoc analysis of a randomized controlled trial in which hypertensive African American patients with LVH and vitamin D deficiency were randomized to receive intensive antihypertensive therapy plus vitamin D supplementation or placebo. We selected patients who had detectable lisinopril (lisinopril group) in plasma using liquid-chromatography/mass spectrometry analysis and compared them to subjects who did not (comparison group) at the one-year follow-up. The pro-fibrotic marker type 1 procollagen C-terminal propeptide (PICP) and the anti-fibrotic markers matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), telopeptide of collagen type I (CITP), and N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) peptide were measured. Sixty-six patients were included, and the mean age was 46.2 ± 8 years. No difference was observed in the number and intensity of antihypertensive medications prescribed in each group. Patients with detectable lisinopril had lower blood pressure than those in the comparison group. The anti-fibrotic markers Ac-SDKP, MMP-1, and MMP-1/TIMP-1 ratio were higher in patients with detectable ACEi (all p < .05). In a model adjusted for systolic blood pressure, MMP-1/TIMP-1 (p = .02) and Ac-SDKP (p < .001) levels were associated with lisinopril. We conclude that ACEi increase anti-fibrotic biomarkers in hypertensive African Americans with LVH, suggesting that they may offer added benefit over other agents in such patients.

Effect of Lower Blood Pressure Goals on Left Ventricular Structure and Function in Patients With Subclinical Hypertensive Heart Disease.

BACKGROUND: Subclinical hypertensive heart disease (SHHD) is a precursor to heart failure. Blood pressure (BP) reduction is an important component of secondary disease prevention in patients with SHHD. Treating patients with SHHD utilizing a more intensive BP target (120/80 mm Hg), may lead to improved cardiac function but there has been limited study of this, particularly in African Americans (AAs). METHODS: We conducted a single center, randomized controlled trial where subjects with uncontrolled, asymptomatic hypertension, and SHHD not managed by a primary care physician were randomized to standard (<140/90 mm Hg) or intensive (<120/80 mm Hg) BP therapy groups with quarterly follow-up for 12 months. The primary outcome was the differences of BP reduction between these 2 groups and the secondary outcome was the improvement in echocardiographic measures at 12 months. RESULTS: Patients (95% AAs, 65% male, mean age 49.4) were randomized to the standard (n = 65) or the intensive (n = 58) BP therapy groups. Despite significant reductions in systolic BP (sBP) from baseline (-10.9 vs. -19.1 mm Hg, respectively) (P < 0.05), no significant differences were noted between intention-to-treat groups (P = 0.33) or the proportion with resolution of SHHD (P = 0.31). However, on post hoc analysis, achievement of a sBP <130 mm Hg was associated with significant reduction in indexed left ventricular mass (-6.91 gm/m2.7; P = 0.008) which remained significant on mixed effect modeling (P = 0.031). CONCLUSIONS: In post hoc analysis, sBP <130 mm Hg in predominantly AA patients with SHHD was associated with improved cardiac function and reverse remodeling and may help to explain preventative effects of lower BP goals. CLINICAL TRIALS REGISTRATION: Trial Number NCT00689819.

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 µg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

Screening and Treatment for Subclinical Hypertensive Heart Disease in Emergency Department Patients With Uncontrolled Blood Pressure: A Cost-effectiveness Analysis.

OBJECTIVES: Poorly controlled hypertension (HTN) is extremely prevalent and, if left unchecked, subclinical hypertensive heart disease (SHHD) may ensue leading to conditions such as heart failure. To address this, we designed a multidisciplinary program to detect and treat SHHD in a high-risk, predominantly African American community. The primary objective of this study was to determine the cost-effectiveness of our program. METHODS: Study costs associated with identifying and treating patients with SHHD were calculated and a sensitivity analysis was performed comparing the effect of four parameters on cost estimates. These included prevalence of disease, effectiveness of treatment (regression of SHHD, reversal of left ventricular hypertrophy [LVH], or blood pressure [BP] control as separate measures), echocardiogram costs, and participant time/travel costs. The parent study for this analysis was a single-center, randomized controlled trial comparing cardiac effects of standard and intense (<120/80 mm Hg) BP goals at 1 year in patients with uncontrolled HTN and SHHD. A total of 149 patients (94% African American) were enrolled, 133 (89%) had SHHD, 123 (93%) of whom were randomized, with 88 (72%) completing the study. Patients were clinically evaluated and medically managed over the course of 1 year with repeated echocardiograms. Costs of these interventions were analyzed and, following standard practices, a cost per quality-adjusted life-year (QALY) less than $50,000 was defined as cost-effective. RESULTS: Total costs estimates for the program ranged from $117,044 to $119,319. Cost per QALY was dependent on SHHD prevalence and the measure of effectiveness but not input costs. Cost-effectiveness (cost per QALY less than $50,000) was achieved when SHHD prevalence exceeded 11.1% for regression of SHHD, 4.7% for reversal of LVH, and 2.9% for achievement of BP control. CONCLUSIONS: In this cohort of predominantly African American patients with uncontrolled HTN, SHHD prevalence was high and screening with treatment was cost-effective across a range of assumptions. These data suggest that multidisciplinary programs such as this can be a cost-effective mechanism to mitigate the cardiovascular consequences of HTN in emergency department patients with uncontrolled BP.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Structure Elucidation and in Vitro Toxicity of New Azaspiracids Isolated from the Marine Dinoflagellate Azadinium poporum.

Two strains of Azadinium poporum, one from the Korean West coast and the other from the North Sea, were mass cultured for isolation of new azaspiracids. Approximately 0.9 mg of pure AZA-36 (1) and 1.3 mg of pure AZA-37 (2) were isolated from the Korean (870 L) and North Sea (120 L) strains, respectively. The structures were determined to be 3-hydroxy-8-methyl-39-demethyl-azaspiracid-1 (1) and 3-hydroxy-7,8-dihydro-39-demethyl-azaspiracid-1 (2) by ¹H- and (13)C-NMR. Using the Jurkat T lymphocyte cell toxicity assay, (1) and (2) were found to be 6- and 3-fold less toxic than AZA-1, respectively.

Structure Elucidation, Relative LC-MS Response and In Vitro Toxicity of Azaspiracids 7-10 Isolated from Mussels (Mytilus edulis).

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7-10, 7-10, were isolated from shellfish and their structures, previously proposed on the basis of only LC-MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4-6, 4-6, and 7-10 were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC-MS(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, 1. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC50 values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure-activity relationships of AZAs.

Isolation, structure elucidation, relative LC-MS response, and in vitro toxicity of azaspiracids from the dinoflagellate Azadinium spinosum.

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (3), AZA34 (4), and AZA35, respectively) in mussels, seawater, and Azadinium spinosum culture. Approximately 700 µg of 3 and 250 µg of 4 were isolated from a bulk culture of A. spinosum, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure-activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of 3 and 4 were found to be 0.22- and 5.5-fold that of AZA1 (1), respectively. Thus, major changes in the carboxyl end of 1 resulted in significant changes in toxicity. In mussel extracts, 3 was detected at low levels, whereas 4 and AZA35 were detected only at extremely low levels or not at all. The structures of 3 and 4 are consistent with AZAs being biosynthetically assembled from the amino end.

Epimers of azaspiracids: Isolation, structural elucidation, relative LC-MS response, and in vitro toxicity of 37-epi-azaspiracid-1.

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

Marine algal toxin azaspiracid is an open-state blocker of hERG potassium channels.

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K(+) current for each AZA analogue was concentration-dependent (IC(50) value range: 0.64-0.84 µM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open-state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [(3)H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC(50) value range: 2.1-6.6 µM). AZA1 did not block the K(+) current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K(+) conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs.

Comparative analysis of three brevetoxin-associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida Panhandle region (USA).

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.

Induction of apoptosis pathways in several cell lines following exposure to the marine algal toxin azaspiracid.

Azaspiracids (AZAs) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although there have been no deaths associated with the AZA toxins, humans exposed to AZAs experience severe gastrointestinal symptoms. This toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Just recently, the AZAs have been shown to be potassium channel inhibitors. This report employed multiple human cell lines [Jurkat T lymphocytes, Caco-2 intestinal cells, and BE(2)-M17 neuroblastoma cells] in characterizing cytotoxicity and pathways of apoptosis. Cytotoxicity experiments were consistent with published literature that has shown that AZA1 is cytotoxic in both a concentration- and time-dependent manner to each cell type tested, with mean EC(50) values ranging between 1.1 and 7.4 nM. Despite the absence of morphological indices indicating apoptosis, caspase-3/7 activity was higher in all cell types treated with AZA1. Furthermore, in T lymphocytes, the most sensitive cell type, the activities of initiator caspase-2 and caspase-10 and concentrations of intracellular cytochrome c were elevated. DNA fragmentation was also observed for T lymphocytes exposed to AZA1-AZA3. Collectively, our data confirm that AZA1 was highly cytotoxic to multiple cell types and that cells exposed to AZA1 underwent atypical apoptosis, possibly in conjunction with necrotic cytotoxicity.

Comparative effects of the marine algal toxins azaspiracid-1, -2, and -3 on Jurkat T lymphocyte cells.

Azaspiracids (AZA) are polyether marine toxins of dinoflagellate origin that accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no monitoring programs or regulatory requirements in the United States for this toxin group. This did not prove to be a problem until June 2009 when AZAs were identified in US seafood for the first time resulting in human intoxications and further expanding their global distribution. Efforts are now underway in several laboratories to better define the effects and mechanism(s) of action for the AZAs. Our investigations have employed Jurkat T lymphocyte cells as an in vitro model to characterize the toxicological effects of AZA1, AZA2, and AZA3. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that AZA1, AZA2, and AZA3 each elicited a lethal response that was both concentration- and time-dependent, with EC(50) values in the sub- to low nanomolar range. On the basis of EC(50) comparisons, the order of potency was as follows: AZA2 > AZA3 > AZA1, with toxic equivalence factors (TEFs) relative to AZA1 of 8.3-fold and 4.5-fold greater for AZA2 and AZA3, respectively. Image analysis of exposed cells using Nomarski differential interference contrast (DIC) imaging and fluorescent imaging of cellular actin indicated that the morphological effects of AZA1 on this cell type are unique relative to the effects of AZA2 and AZA3. Collectively, our data support the growing body of evidence suggesting that natural analogues of AZA are highly potent and that they may have multiple molecular targets.