Sunday, September 24, 2017

Red Tides; they're keeping this one under wraps; turn to God now; the end is near

You know I had to try several times to get info on this very dangerous worldwide phenomenon that threatens the safety and security of the world.

Review of Florida Red Tide and Human Health Effects

Abstract

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue—one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.
Keywords: Florida red tide, red tide, neurotoxic shellfish poisoning, NSP, brevetoxins, harmful algal bloom, HAB, Karenia brevis, shellfish poisoning, respiratory irritation, marine toxin diseases, neurotoxic fish poisoning

1.0 Introduction

Florida red tides are predominantly associated with the blooms of the toxic dinoflagellate, Karenia brevis (K. brevis), formerly known as Gymnodinium breve and Ptychodiscus brevis. K. brevis produces a group of potent natural neurotoxins, the brevetoxins (i.e. PbTx or Ptychodiscus toxins), which can cause illness and mortalities in fish, seabirds, and marine mammals. Humans are susceptible to the effects of brevetoxin exposure, and public health surveillance activities have documented cases of intoxications from eating contaminated seafood and many respiratory complaints from inhaling contaminated aerosols (Kirkpatrick et al., 2004a).
Florida red tide blooms have been documented on the Florida west coast since the 1800s. More recently, Florida red tides have spread as far as the eastern coast of Mexico and have been entrained in the Gulf Loop, the current that brings Gulf waters to the shores of North Carolina. Other brevetoxin-producing dinoflagellate blooms have been identified in diverse geographic locations worldwide, including New Zealand, Australia and Scotland (Baden and Fleming 2007; Hernandez Becerril et al., 2007; Haywood et al., 2004; Kirkpatrick et al., 2004a; Nozawa et al., 2003; Steidinger et al., 1983).
In the 1980s and 1990s, there was increased interest in and research activity on harmful algal blooms (HABs). Much of this interest was driven by media attention on the discovery of several new HAB organisms purportedly associated with animal and human exposures and health impacts (e.g. Pseudo-nitzschia, Pfiesteria, and the phytoplankton producing the newly discovered toxins, the Azaspiracids) (Abraham and Baden 2006; Backer et al., 2003a; Backer et al., 2005a; Backer and Fleming 2008; Fleming et al., 2001; Fleming et al., 2004; Okamoto and Fleming 2005; Twiner et al., 2008; Zaias et al., 2010). There were also concerted efforts by the HAB research and response community to increase national and international attention on the apparent increase in HABs and the resulting increased risk for human exposure and subsequent adverse health effects (e.g. National HAB plan at http://www.esa.org/HARRNESS/). These activities lead to increased funding and interest in HAB research (e.g. the ecology and oceanography of harmful algal blooms [ECOHAB] http://www.whoi.edu/science/B/redtide/nationplan/ECOHAB/ECOHABhtml.html).
Over the past decade, there has been an intensive interdisciplinary and inter-agency research program focused on the possible exposures and health effects in humans and other animals from the Florida red tide toxins, particularly the aerosolized toxins (Abraham and Baden 2006; Backer et al., 2003a; Backer et al., 2005a; Fleming et al., 2004; Fleming et al., 2005a; Red tide research group 2002; Zaias et al., 2010). The findings of this particular research program are summarized below, with particular emphasis on the implications for human health.

2.0 Organisms

In the past decade, through the use of new technologies, it has become clear that K. brevis is only one of several different species of the Genus, Karenia, found throughout the world’s oceans. Blooms in the Gulf of Mexico may contain both K. brevis and K. mikimotoi (another brevetoxin-producing Karenia species) (Haywood et al., 2004). Other research has demonstrated varying brevetoxin production among the Karenia species, and even among individual K. brevis organisms. There have also been major advances in understanding the genomics of these dinoflagellates, such as the identification of the toxin-producing PKS genes, exploration of the impact of environmental change (e.g. temperature, light/dark cycles, etc) on gene expression, and the appreciation of the apparently unique complexity of the Karenia genome (Lidie et al., 2005; Monroe et al., 2008; Rein and Snyder 2006).
Despite what has been learned in the past few decades, there remains ongoing controversy concerning the sources and factors contributing to the bloom behavior of dinoflagellates, including K. brevis. The actual life cycle of K. brevis is still undefined, especially the location or existence of resting cysts. However, the major controversy has centered on the ability of anthropogenic change to influence K brevis bloom dynamics In particular, the relative importance of the role of nutrients (e.g. nitrates, phosphorus, silica, and iron) from coastal rivers, non-point coastal sources, or atmospheric deposition in initiating and/or sustaining K. brevis blooms is currently an important research and environmental policy topic (Brand and Compton 2007; Olascoaga et al., 2006; Walsh J et al., 2006).

3.0 Toxins

There has been an explosion of research on brevetoxins research over the past decade due to increased scientific and public health interest, and the potential to apply a range of new technologies. The brevetoxins (M.W. ~900) are lipid soluble, cyclic polyethers. In biological systems, they act to open voltage gated sodium (Na+) ion channels in cell membranes, leading to Na+ influx into the cell (Baden and Fleming 2007; LePage et al., 2003; Mattei et al., 2008; Twiner et al., 2007). There are over 10 different brevetoxins isolated in sea water blooms and K. brevis cultures in the laboratory, as well as multiple analogs and derivatives from the metabolism of shellfish and other organisms (Baden et al., 2005; Baden and Fleming 2007; Campbell et al., 2004; Michelliza et al., 2004 and 2007; Satake et al., 2008 and 2009). Recently, several laboratories have successfully synthesized brevetoxins de novo (Crimmins et al., 2009; Fuwa et al., 2006; Kuranaga et al., 2009). During Florida red tide blooms, the major brevetoxin produced is PbTx-2, along with lesser amounts of PbTx-1, PbTx-3, and other brevetoxin analogs (Cheng et al., 2004; Cheng et al., 2005a; Cheng et al., 2005b; Hardman et al., 2004; Lamberto et al., 2004; Pierce et al., 2003; 2005; 2008).
One of the most exciting discoveries of the last decade has been the identification of brevenal, a brevetoxin antagonist, in both K. brevis laboratory culture and in the environment during K. brevis blooms. This is apparently the first documented case of a toxin-producing organism also producing its own antagonist. Brevenal is produced by K. brevis in significant amounts, particularly during bloom senescence, and it acts at a different receptor site on nerve cells than the brevetoxins. Other brevetoxin analogs with varying degrees of antagonism or brevetoxin-like characteristics have also been identified (Abraham et al., 2003; Abraham et al., 2005a and 2005b; Bourdelais et al., 2002; Bourdelais et al., 2004a and 2004b; Bourdelais et al., 2005; Potera 2007).
From the point of view of human exposure and health, the brevetoxins are tasteless, odorless, and heat and acid stable. Thus, these toxins cannot be easily detected, nor can they be removed by food preparation procedures (Backer et al., 2003a; Backer et al., 2005a; Backer and Fleming 2008; Baden and Fleming 2007). Thus, the normal warning mechanisms (e.g. bad taste) or other protections (e.g. cooking contaminated seafood) are useless, and public health protection must focus on preventing human exposure (i.e. primary prevention).
Over the past decade, again thanks to the application of new technologies, major advances have been made in the detection of brevetoxins in a range of substrates, including seawater, air, seafood, and various animal and human clinical specimens (Dechraoui 2005; Dickey et al., 2004; Flewelling et al., 2005; Naar et al., 2007; Plakas et al., 2008; Poli et al., 2007; Weidner et al., 2004; Woofter et al 2003, 2005a and b, 2007). In particular, the creation, development and application of a new brevetoxin ELISA to all of these substrates, coupled with significant improvements in the detection limits of more traditional toxicologic analyses (e.g. Liquid Chromatography Mass Spectrometry (LCMS)), have allowed researchers and regulators to identify brevetoxins at very low levels in multiple environments and in a range of substrates (Naar et al., 2002 and 2004). This improved detection ability has been particularly important in advancing research to document exposures to brevetoxin-contaminated aerosols generated during Florida red tides and to identify the associated health effects in animals and humans. Specifically, using this highly sensitive ELISA, brevetoxins (particularly PbTx2 and 3, as well as brevenal) have been found in seawater and aerosols during active K. brevis blooms, as well as during non bloom periods (although at much lower levels).
In addition to establishing the concentrations of brevetoxins seen during a Florida red tide bloom (ranging from 15–90 mg/m3), the particle size of the brevetoxin aerosol has been characterized. The particles have a geometric mean of approximately 8–9 µ. This is important information in terms of the potential for respiratory effects of brevetoxin aerosols in humans. Particle size needs to be less than 5 µ to enter the lower airway; therefore, with a geometric mean of 8–9 µ, only 10–20% of these particles are small enough to enter the human lung (Cheng et al., 2004; Cheng et al., 2005a; Cheng et al., 2005b; Pierce et al., 2003; 2005; 2008). Using the same air sampling technologies, brevetoxin aerosols have been demonstrated to travel as much as a mile inland from coastal areas during an active Florida red tide, particularly when there are strong onshore winds (Kirkpatrick et al., 2010).
In addition to its application for aerosol analysis, improvements in brevetoxin detection and measurement have lead to the discovery of measurable levels of toxin in fish, both in the internal organs and in the filets that might be eaten by people. This creates the new possibility that there is an additional disease, “brevetoxin fish poisoning” (not just shellfish poisoning) that could effect marine mammals and people as discussed below (Flewelling et al., 2005; Kirkpatrick et al., 2009a; Naar et al., 2007).

4.0 Cellular processes

Cellular effects associated with both natural and experimental exposure to brevetoxins have been observed in the immune system of many species, although the mechanisms of action of brevetoxin exposure on immune cells and immune competence are not well understood. The number and variety of mediators, critical checkpoints, and key regulators in the immune system are vast, and brevetoxin may impact any one of these pathways individually or in combination. Full characterization of cellular consequences of brevetoxin exposure is critical to fully understand the impact of recurrent red tide events on human health.
Several potential mechanisms for brevetoxin immunotoxicity have been suggested, including the inhibition of cathepsin active sites (Katunuma et al., 2003; Sudarsanam et al., 1992); apoptosis (Bossart et al., 1998; Sayer et al., 2005; Walsh et al., 2008; Murrell and Gibson, 2009; 2010); the release of inflammatory mediators (Bossart et al., 1998; Murrell and Gibson 2010); effects on cell cycle (Han et al., 2003; Murrell and Gibson, 2009; Sayer et al., 2006; Walsh et al., 2005; 2008); and oxidative stress (Radwan and Ramsdell, 2006; Walsh et al., 2009). Brevetoxin exposure has been shown to have the potential to impair the immune system of many species, including manatee (Bossart et al., 1998; Walsh et al., 2005), cormorant (Kreuder et al., 2002), rat (Benson et al., 1999; 2004a and b; 2005) and loggerhead sea turtle (Walsh et al., 2010). Demonstrated effects resulting from brevetoxin exposure include: reduced phagocytosis (Benson et al., 1999); decreased plaque-forming ability (Benson et al., 1999; 2004a and b); and decreased lymphocyte proliferation (Walsh et al., 2005). Levels of lysozyme were found to be elevated in rescued loggerhead sea turtles (Walsh et al., 2010). In vitro experiments have demonstrated possible DNA damage (Murrell and Gibson, 2010; Sayer et al., 2005); chromosomal aberrations (Sayer et al., 2006); and effects on cellular growth (Han et al., 2003; Murrell and Gibson, 2009; Sayer et al., 2006; Walsh et al., 2008). Other immune system effects include mast cell degranulation (Hilderbrand et al., 2010) and histamine release (Abraham et al., 2005), cellular effects which may contribute to observed airway responses following the inhalation of aerosolized brevetoxins. Production of the pro-inflammatory cytokine, IL-6, was increased at both the protein (Hilderbrand et al., 2010) and gene (Murrell and Gibson, 2010) level in response to brevetoxin exposure. Several other cytokine genes with roles in pathogenesis of respiratory diseases were also shown to be increased in Jurkat E6-1 cells in response to in vitro brevetoxin exposure (Murrell and Gibson, 2010).
Apoptosis as potential mechanism of brevetoxin immunotoxicity was suggested based on the presence of interleukin-1 converting enzyme in lymphocytes and macrophages in manatee tissues collected during an epizootic (Bossart et al., 1998). DNA damage in human lymphocytes treated with brevetoxins in vitro (Sayer et al., 2005) supports apoptotic effects. Apoptosis, as measured by activity of caspase-3, was reported in a cell line (Jurkat E6-1) exposed to PbTx-2 and PbTx-6, but not when exposed to PbTx-3 (Walsh et al., 2008). Murrell and Gibson (2009) also demonstrated apoptosis occurring in brevetoxin-treated Jurkat cells through an increase in caspase 3/7 activity and activation of poly (ADP-ribose) polymerase (PARP), processes which were toxin-congener dependent, again with PbTx-3 failing to induce apoptosis. Several genes involved in apoptotic processes were affected by in vitro brevetoxin exposure in Jurkat cells (Murrell and Gibson, 2010).
Several studies have indicated that oxidative stress may play a role in the cellular response to brevetoxins. Glutathione depletion, an indication of oxidative stress, resulted in a U-937 human monocyte cell line treated with PbTx-2 (Walsh et al., 2009). Observations of DNA strand breaks (Sayer et al., 2005) and chromosomal aberrations (Sayer et al., 2006) are also consistent with oxidative stress. Brevetoxins have been shown to proceed through cytochrome P450 metabolic pathways, which may lead to oxidative damage. Evidence for cytochrome P450 involvement includes the metabolism of PbTx-2 by rat hepatocytes (Radwan and Ramsdell, 2006) and the U-937 human monocyte cell line (Walsh et al., 2009), and following treatment with cDNA-expressed rat cytochrome P450 enzymes (Radwan et al., 2005). The systemic administration of PbTx-2 to rats (Radwan et al., 2005) also demonstrated brevetoxin metabolism through cytochrome P450 pathways. Such metabolic processes can generate nucleophilic intermediates with the potential to bind DNA, and may have led to the brevetoxin-nucleic acid adducts detected in rat lung cells following both in vitro and in vivo exposure (Radwan and Ramsdell, 2008). Leighfield et al. (2009), however, reported that neither PbTx-2 nor the epoxide (PbTx-6) showed mutagenic potential. Some genes related to DNA damage, however, were increased in expression in Jurkat cells exposed to brevetoxin (PbTx-2) (Murrell and Gibson, 2010).

5.0 Animals

Significant die-offs of marine mammals, seabirds, and other animals throughout the 1990s and early 2000s enhanced awareness of the impacts of Florida red tides, and led to substantial increases in the resources available to support relevant interdisciplinary research (Kirkpatrick et al., 2004; Kreuder et al., 2002; Van Dolah et al., 2003; Zaias et al., 2010). In particular, the deaths of a significant population of the highly endangered Florida manatee during the prolonged 1996 Florida red tide focused attention on the potential health impacts for both animals and humans, particularly those associated with inhaling aerosolized toxins (Bossart et al., 2002; 2003a; 2003b). Another important finding demonstrating the impacts of brevetoxins on animals involved a major dolphin die-off in the early 2000s. Although not temporally associated with an active Florida red tide bloom, the cause of death was exposure to brevetoxins via the food web. Fish found in the dolphins’ stomachs tested positive for brevetoxins, particularly in the organs but also in the muscle. This episode raised the possibility of “brevetoxin fish poisoning” in humans and other animals who consumed whole fish contaminated with brevetoxins (Flewelling et al., 2005; Kirkpatrick et al., 2009a; Naar et al., 2007).
Recent laboratory studies in animals have been particularly important in exploring exposure and toxicity mechanisms, validating brevetoxin exposure, and demonstrating biological plausibility and possible mechanisms of action for the health effects reported in human studies (described below). Short and long term exposures of rodents (rats and mice) to aerosols containing brevetoxin have not demonstrated the same level of toxicity as seen in humans and other animal models. However, rodent studies have shown that aerosolized exposure to brevetoxins can lead to rapid systemic distribution, particularly to the neurologic system, implying potential adverse neurologic health impacts with respiratory exposure to aerosols (Benson et al., 2004a and 2004b; Benson et al., 2005a; Tibbetts et al., 2006). For example, exposing mice to aerosols containing brevetoxins (and exposing fish to water containing brevetoxins) caused changes in vestibular and auditory nerve function (Benson et al., 2005b; Lu and Tomchik 2002). Long term exposures of rodents to aerosolized brevetoxins have demonstrated immune dysfunction, including delay of viral clearance and possible enhancement of the pathogenicity of influenza A (J Benson, Lovelace Respiratory Research Institute, personal communication). Rodent studies have also been important in demonstrating that brevetoxins delivered in aerosols are not teratogenic in multi-generational exposure studies (Benson et al., 2006).
The sheep model of asthma has served as an important tool for the exploration of possible health effects from aerosolized brevetoxin exposures. The highlight of this model is that the sheep respond to brevetoxin exposures at levels similar to those experienced by humans at the beach during a Florida red tide bloom (Abraham and Baden 2006; Abraham et al., 2003; Abraham et al., 2004; Abraham et al., 2005a and b; Abraham et al., 2009; Zaias et al., in press). This model has demonstrated that both asthmatic and non asthmatic sheep react with significantly decreased respiratory function and experience dose-dependent airway hypersensitivity after exposure to very small concentrations of aerosolized brevetoxins (~10 pg/ml of PbTx-2 or PbTx-3, the two main toxins found in the air during a K. brevis bloom). In the asthmatic sheep, these effects are larger and last longer, particularly when there has been exacerbation of the asthma prior to the brevetoxin exposure. Chronic exposures in sheep demonstrate reduced function of alveolar macrophages, suggesting immune dysfunction (Zaias et al in press). This same model system has been important in the exploration of the pathogenesis and binding of many different types of brevetoxins and their analogs, including the new antagonist brevenal, since it allows for precise exposure delivery and effect measurement.

6.0 Humans

Humans can be exposed to brevetoxins through food, water, and air (Backer et al., 2003a; Backer et al., 2005a; Backer and Fleming 2008; Fleming et al., 2001; Fleming et al., 2002; Okamoto and Fleming 2005). Until recently, the health effects associated with exposure to Florida red tide have been driven primarily by anecdote and case report, as well as the evidence described above from wild marine mammal illnesses and deaths. It is only in the past decade that interdisciplinary epidemiologic research has been applied to the exposures and health effects of Florida red tide and its toxins.

6.1. Consumption of contaminated seafood

The traditional illness associated with exposure to Florida red tide and its toxins through the consumption of contaminated shellfish is neurotoxic shellfish poisoning (NSP). The assumption has been that this is a relatively rare disease due to the stringent monitoring and timely closure of toxin-contaminated shellfish beds in the Gulf of Mexico. However, a recent comprehensive Review by Watkins et al., (2009) found that, this illness is likely to be misdiagnosed, and is probably more common than previously thought, particularly among visitors and subpopulations not informed of shellfish bed closures or shellfish harvesting bans. Based on a review of emergency room cases in Florida, it is clear that NSP can be a severe acute disease with emergency room and intensive care required during the first hours, and, in severe cases days, to prevent respiratory failure (Abraham A et al., 2008; Watkins et al., 2009). Even with a severe acute illness, victims are usually discharged from the hospital within days; there is almost nothing known about the subchronic or chronic sequelae of an acute NSP episode. Furthermore, nothing is known about the possible health effects of long term very low level exposures from eating shellfish with low levels of contamination over a long period of time.
With regards to the possible new illness of “brevetoxin fish poisoning,” it is not known if there are human cases of illness associated with eating brevetoxin-contaminated finfish. Nevertheless, evaluation of emergency room admissions for gastrointestinal illnesses during an active Florida red tide and again when there was not an active bloom demonstrated significantly increased gastrointestinal illness emergency room admissions during the active Florida red tide period (Flewelling et al., 2005; Kirkpatrick et al., 2009a; Naar et al., 2007; Perez Linares et al., 2009).



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