Environmental Toxicology and Pharmacology 19 (2005) 401406

Risk of human exposure to paralytic toxins of algal origin

M.C.C. Bator ua, , E. Diasb , P. Pereirab , S. Francab e
b a Faculty of Pharmacy, Laboratory of Toxicology, Av. Prof. Gama Pinto, 1649-043 Lisbon, Portugal Laboratory of Microbiology and Ecotoxicology, National Health Institute, Dr. Ricardo Jorge, Lisbon, Portugal

Abstract The most signicant neurotoxins produced by harmful algal blooms (HABs) are paralytic shellsh toxins (PSTs) found in shellsh and freshwater. Human exposure to neurotoxins through the food consumption represents a severe hazard to human health and the exposure through contaminated water represents an added risk often difcult to recognize. Furthermore, there is an insufcient knowledge of toxicokinetics of these complex toxins produced by HABs. If human acute exposure occurs, the diagnosis of intoxication is typically based upon symptomatology and analysis of shellsh tissue by mouse bioassay, HPLC-FLD analysis and mouse neuroblastoma assay. However, the health risks due to chronic exposure should also be considered and its prevention could be reached with a better understanding of sub-lethal doses of these toxins. In this context, information required for development of a diagnostic protocol should include knowledge about toxicokinetics and toxicodynamics of these neurotoxins. We emphasise the importance of research on biomarkers to prevent, predict and diagnose acute and chronic human exposure to PST. 2005 Elsevier B.V. All rights reserved.
Keywords: Paralytic shellsh toxins; Neurotoxicity; Human exposure; Toxicokinetics; Biomarkers; PST monitoring

1. Introduction Phytoplanktonic organisms are natural components of water environments that, under favourable environmental conditions (such as light, temperature, oxygen and nutrients) or due to anthropogenic action (fertilizers input) may proliferate at a high rate (Hallegraeff, 1993). When particular species dominate the phytoplanktonic community, they accumulate and may form a visible and dense biomass lm in water surface commonly known as algal blooms. This phenomenon is called harmful algal bloom (HAB), namely when the proliferating species produce toxins (phycotoxins) that may be a threat to human health. In the sea HABs are mainly caused by dinoagellates (Anderson, 1994) while in freshwater toxic blooms are due to cyanobacteria (Carmichael, 1994). Some of these phycotoxins are neurotoxins (Table 1), and are usually denominated by the effects that they cause or by the name of producer specie. In marine environments,

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dinoagellates are responsible for the occurrence of paralytic shellsh toxins (PSTs), produced by several species, and brevetoxins, produced only by Karenia brevis. Also some species of the bacillariophycea pseudo-nitzschia, are capable of producing the neurotoxin, domoic acid, responsible for amnesic shellsh poisoning (ASP). Crustaceous, mollusc and sh that feed from phytoplankton accumulate those toxins, transmitting them to humans through food chain (Hallegraeff, 1993). In freshwater, several cyanobacterial species may also produce PST and anatoxins (rstly attributed to Anabaena sp.) that can also accumulate in the food chain or be transmitted to humans through the ingestion of contaminated water (Negri and Jones, 1995; Chorus et al., 2000). Paralytic shellsh poisoning (PSP) is a very well known human syndrome caused by an acute intoxication after the ingestion of shellsh contaminated with paralytic toxins. PSP symptoms in mammals ranges from nausea, vomiting, numbness of the lips and tongue and muscle paralysis to death by cardio-respiratory arrest (Price et al., 1991). The most current diagnostic methodologies are the clinical history and the toxin analysis in seafood. Nevertheless, no stud-

1382-6689/$ see front matter. 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.12.002

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Table 1 Neurotoxins produced by phytoplanktonic species Environment Sea water Producer organism Dinoagellates Gymnodinium catenatum Alexandrium spp. Karenia brevis Diatoms Pseudo-nitzschia spp. Freshwater Cyanobacteria Anabaena spp. Aphanizomenon spp. Cylindrospermopsis sp. Lyngbya sp. Neurotoxins PST PST Brevetoxins ASP Intoxication vehicle Molluscs Crustaceans Fish

Anatoxins, PST Anatoxins, PST PST PST

Water Molluscs Crustaceans Fish

ies on biomarkers to control and/or identify intoxications due to acute or chronic human exposure to these natural contaminants have been reported.

2. PST occurrences in marine and freshwater PSP has been considered a serious risk for shellsh consumers for many centuries ago. The rst description of paralytic shellsh poisoning dates from 1793 and in 1927 a serious epidemic intoxication in San Francisco was ascribed with certainty as paralytic shellsh poisoning. Dinoagellates were associated with that intoxication and the Californians PSP prevention program was implemented (Price et al., 1991). In Europe, similar programs are mandatory since the 1990s (Van Egmond et al., 1993). Also, globally many programs have been established in several susceptible regions (Hallegraeff, 1993). There are many reports of PSP worldwide associated with human severe intoxication and death (Table 2). In 1993, the estimated incidence was 2000 cases per year with 15% of mortality (Hallegraeff, 1993). Nowadays, death by PSP episodes occurred mainly in those countries where prevention programs were not still adopted. In Portugal, the most important episode of PSP occurred in 1946 when 100 people was intoxicated and 6 patients died after the consumption of PST contaminated shellsh from Obidos Lagoon (Franca, 1991). After the implementation of monitoring program in 1986 the few reported cases were asTable 2 Reported human paralytic shellsh poisoning Country/region North America UK Guatemala Morocco Norway Portugal Chile East Timor Year 19001989 1968 1987 1994 19011992 1946/1994 1972/98 2002 No. of intoxications 1399 78 187 77 32 132 329 1 No. of deaths 90 26 4 2 7 23 1

sociated with the disrespect of shellsh consumption prohibition (Carvalho et al., 1998). More recently (in the past decade), PST production by cyanobacteria was also found in freshwater supplies worldwide (Sivonen and Jones, 1999). In Portugal, for example, three occurrences of cyanobacterial blooms associated with PST were detected between 1995 and 1999 (Fig. 1). Those were attributed to different species of Aphanizomenon spp. (Pereira et al., 2000, 2001).

3. PST toxicity PST are a group of carbamate alkaloid compounds that can be divided in three classes according to their degree of sulphatation (Oshima, 1995) that, in turn, are related with their specic toxicities (Table 3). The class of non-sulphated compounds includes the most potent toxins, like saxitoxin that presents an LD50 after an i.p. injection in mice of only 10 g/kg bw, and an oral LD50 of 263 g/kg bw (WHO, 1984). The singly sulphated gonyautoxins present a moderate to high toxicity and the double sulphated C-toxins are the less toxic class. PST act by reversible binding to the sodium ion channels of nerve membranes (Catterall, 1980) (Fig. 2), blocking neuronal transmission and causing a generalised nerve dysfunction (Easthaugh and Shepherd, 1989). Despite the mechanism of toxicity of saxitoxin and its derivatives is known at molecular and cellular level, there is not much research about their toxicokinetics in mammals and the few available data are still inconclusive. Studies with cats administrated orally with gonyautoxins 23 (Andrinolo et al., 2002a) suggest that PST absorption occurs exclusively via intestinal epithelium and the excretion mainly involves glomerular ltration. In vitro assays with rat and human cell lines pointed out to the role of an active transport system involved in PST intestinal absorption (Andrinolo et al., 2002b). In a previous study, these authors reported saxitoxin (i.v. administration in cats) distribution through liver, spleen and central nervous system, indicating that toxin is capable of crossing the bloodbrain barrier (Andrinolo et al., 1999). In this study, they also con-

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Fig. 1. Occurrence of cyanobacterial blooms associated with paralytic shellsh toxins in Portuguese freshwater reservoirs.

Table 3 Structure and specic toxicity of paralytic shellsh toxins (adapted from Oshima, 1995)

Toxin STX neoSTX dcSTX GTX1 GTX2 GTX3 GTX4 GTX5 GTX6 C1 C2 C3 C4

R1 H OH H OH H H OH H OH H H OH OH

R2 H H H H H OSO3 OSO3 H H H OSO3 H OSO3

R3 H H H OSO3 OSO3 H H H H H H OSO3 H

R4 CONH2 CONH2 H CONH2 CONH2 CONH2 CONH2 CONHSO3 CONHSO3 CONHSO3 CONHSO3 CONHSO3 CONHSO3

Specic toxicity (MU /mol) 2483 2295 1220 2468 892 1584 1803 160 180 15 239 33 143

Fig. 2. PST plugs and blocks the voltage-dependent Na+ channel in neurons (adapted from Boelsterli, 2003).

cluded that STX was only eliminated by urine and that mammals cannot metabolise this toxin. However, Llewellyn et al. (2002) reported a case study where he compares the PST contents in the gut and urine of an intoxicated victim, detecting an alteration in PST prole, which reveal a decrease in toxicity of urine saxitoxin derivatives. Other assays performed in rats (Stafford and Hines, 1995) and in intoxicated humans (Gessner et al., 1997), conrmed that the main route of excretion of saxitoxin derivatives is the urine, being 50% excreted as unchanged saxitoxin compounds. Thus, there are

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still many opened questions related with PST toxicokinetics that should be investigated in order to contribute to a better diagnosis and treatment of human poisoning.

4. Monitoring and prevention of human exposure to PST In aquatic environments, PST enter in the food web via lter feeding molluscs and crustaceans, that can present a serious health hazard to higher predators such as sh, birds, marine mammals and humans (Bretz et al., 2002). Some studies demonstrated that biota, according to their trophic level, may present marked interspecic differences in PST sensitivity. Besides, toxin biotransformation may vary greatly among species, which may lead to changes in net toxicity and in toxin accumulation. However, it has also been reported that some lter-feeding shellsh as the bivalve molluscs, and some species of crustaceans such as crabs, accumulate PSTs without signicant biochemical changes. Knowing that the majority are sedentary species, some of those organisms are reported as PSP toxin vectors and considered as good indicators to be used in monitorization programs whereas others undergo extensive toxin biotransformation and can be useful to predict the timing and duration of blooms (Bricelj and Shumway, 1998). Current technology and the timely sampling of outbreakrelated specimens offer the opportunity for a rapid increase in the control and prevention of human exposure to these toxins. There are several analytical and biological methods to detect the toxins in shellsh. Mouse bioassay is the ofcial method for PST monitoring in shellsh (AOAC, 1984). Despite its relative high specicity, other biological systems such as mammalian neuroblastoma cell lines (Louzao et al., 2003) have been developed in order to avoid animal use and to improve the detection limit. Biological methods quantify toxicity as saxitoxin (STX) equivalents but they do not distinguish between different PST components. Thus, to identify individual PST and to quantify each PST components, several HPLC methods with uorescent detection have been developed (Oshima, 1995; Lawrence and Niedzwiadek, 2001). Studies on imunoenzymatic methods to detect PST (Metcalf and Codd, 2003), like ELISA, have presented some difculties and optimisation processes are still required to its implementation. Mass spectrometry (MS) and LCMS have also been used (Harada et al., 1999). Although HPLC has been found very satisfactory, it is limited by the low availability of analytical standards for all PST variants, which are necessary for peak conrmation. Restriction in saxitoxin and derivatives manufacture and use are due to the fact that those compounds are listed by the Organization for the Prohibition of Chemical Weapons (OPCW) as a Schedule 1 (Andrinolo et al., 2002a). There is no antidote for paralytic shellsh poisoning and the only therapeutic actions are to carry out supportive therapy to the patients and adopt rigorous preventing measures.

Monitoring programs to assess toxin levels in shellsh and banning the shellsh harvesting in suspected areas, according to the safety value of 80 g STX eqv/100 g of shellsh tissue, had proved to be of a great value. Similar preventive measures should be applied to freshwater systems, such as the monitoring of toxins in water, the prohibition of bathing in recreational areas and change the drinking water source. In fact, if the risk of PSP can be controlled in shellsh, the same is not applied to bathing and drinking water. In this case, no mandatory limit or even a guideline value for PST in drinking water was adopted (Chorus et al., 2000; Duy et al., 2000). Despite the severe acute effects and the serious threat to public health, there are some difculties in assessing the risk of human exposure to PSTs through freshwater. This is due, in part, to the following gap knowledges. 4.1. Dynamics of PST production by cyanobacteria and toxin transmission to humans through the food chain Regardless the numerous reports on the occurrence of neurotoxic blooms worldwide, the available data still not enables the complete clarication of differences in toxin production ability by cyanobacterial species and to distinguish between toxic and non toxic strains; of environmental factors that inuence toxin production; of mechanisms of toxin release into extracellular medium and the toxin stability in water. There is also scarce knowledge regarding the accumulation/transformation of toxins through the several trophic levels. Therefore, it is still not possible to predict HAB occurrences, to estimate the human exposure doses to cyanobacterial neurotoxins in water and, consequently, to identify and prevent the risk at the origin. 4.2. Efciency of water treatment systems in removing toxins from water supplies Algaecides, such as copper sulphate, are commonly used to control cyanobacterial growth in freshwater reservoirs. However, if cell numbers are high, the massive destruction of cyanobacterial biomass by cell lyses causes the release of toxins from intracellular medium to water (Hrudey et al., 1999). Besides, the dissolved toxins are not effectively removed from water by the conventional water treatment systems (coagulation, sedimentation, ltration, chlorination) (Duy et al., 2000). This is particularly important in small water reservoirs, where high toxin levels may accumulate and persist for long periods (Dias, 2001). However, only few studies have been conducted in order to develop alternative methodologies for removal of cyanotoxins. 4.3. Effect of repeated exposure to low PST doses It should be emphasised that populations served by water resources with a high frequency of neurotoxic blooms may be continuous exposure to low doses of PST. No studies at the moment have been conducted in order to evaluate the health

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impact of this situation. Furthermore, effects of animal and human repeated exposure to PST doses are still not identied. 4.4. Epidemiological data on human PSP in freshwater sources Absence of any report on human acute intoxication by PST through freshwater environments also restrict risk assessment of PSP caused by toxic cyanobacteria. Contrarily, there is some information regarding the effects of other cyanobacterial toxins (hepatotoxins) on humans after the ingestion of raw water (Chorus et al., 2000; Duy et al., 2000). There are also reports of animal poisoning with PST due to the ingestion of water from reservoirs after a neurotoxic bloom occurrence (Negri et al., 1995). Despite all of these gaps, some eld and laboratory studies have revealed that high proportions of toxins may be found in freshwater, particularly after a toxic bloom collapse. During cell growth, toxins are mainly restricted to intracellular compartment, but as the cells become senescent, toxins are massively released to water (Negri et al., 1997; Dias et al., 2002), where they may persist for 12 months (Dias, 2001). In this situation, there is not any sign of cyanobacterial contamination and if no warning of health risk is signalled, bathers do not avoid water contact, despite they may be exposed even to high PST concentrations. An in vitro study with toxic Aphanizomenon strain cultures revealed that extracellular PST levels might reach 2700 g STX eqv/L (Dias et al., 2002). Assuming that in natural environment the same concentrations may be expected, an accidental ingestion of 100 mL of PST contaminated water may be sufcient to cause mild PSP symptoms. According to Price et al. (1991), 200500 g STX eqv cause mild PSP symptoms, 5002000 g STX eqv cause moderate PSP symptoms and doses above 2000 g STX eqv are lethal. Besides raw water, high proportions of PST may also be found in water for human consumption given the ineffectiveness of water treatment plants in removing cyanotoxins. Thus, unless signicant dilution occurs and/or effective operating techniques are used to remove dissolved toxins from contaminated water, a real risk of human exposure to PST through freshwater must be considered. Although ecological, epidemiological and toxicological data still not enable the establishment of a PST guideline for freshwater, there was recently a proposal for an alert level of 3 g STX eqv/L for drinking water (Llewellyn et al., 2001), which emphasise, once more, the potential health risk associated with cyanobacterial neurotoxic blooms.

ing of PST in shellsh by mouse bioassay (and toxin identication by HPLC-FLD) and the shellsh harvesting prohibition if toxin levels exceed 80 g STX eqv/100 g edible tissue. Toxins responsible for this poisoning also occur in freshwater environments but, in this case, only few preventive measures may be adopted. At the moment, there are insufcient scientic data that enables the implementation of regulatory policy. Preventive measures include the monitoring of toxic cyanobacteria and PST in freshwater, the changing of raw water source for production of drinking water and the prohibition of bathing in contaminated recreational water. The goal of all these measures is the prevention of acute human intoxication with PST. However, in many cases the occurrence of neurotoxic species is very frequent, even if the cell densities may not reach the concentrations that can trigger an acute symptomatology. Thus, the development of specic and sensitive biomarkers of PST in biological samples would contribute to: alert to the risk of human exposure to repeated PST low doses; correlate the biomarker levels and the probable development of delayed neurotoxic effects; conrm an acute PSP for immediate curative action, since not all symptoms and signs are highly specic to this intoxication; follow-up the patients recovery through the analysis of toxin levels, prole and detoxication compounds; investigate the toxicokinetic and toxicodynamic of saxitoxin and derivatives; re-evaluate the mandatory limit in shellsh and establish a guideline value for PST in freshwater. Finally, the search for biomarkers of exposure and effect may be useful to predict and prevent human exposure to PST and contribute to the diagnosis and the recovery follow-up of human acute or chronic PSP.

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