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Pectenotoxins — an issue for public health
Environment International 27 (2001) 275 – 283 www.elsevier.com/locate/envint
Pectenotoxins — an issue for public health A review of their comparative toxicology and metabolism Vanessa Burgessa,b,*, Glen Shawb,1 a
National Research Centre for Environmental Toxicology (NRCET), 39 Kessels Road, Coopers Plains, Brisbane, QLD 4108, Australia b School of Public Health, Griffith University, Logan Campus, Brisbane, QLD 4111, Australia Received 8 December 2000; accepted 18 May 2001
Abstract Pectenotoxins (PTXs) are a group of toxins associated with diarrhetic shellfish poisoning (DSP) and isolated from DSP toxin-producing dinoflagellate algae. Consumption of shellfish contaminated with PTXs has been associated with incidences of severe diarrhetic illness resulting in hospitalisation. Concern has been raised for public health following the discovery that these toxins are not only hepatotoxic and can cause diarrhetic effects in mammals, but that they are potently cytotoxic to human cancer cell lines and have been found to be tumour promoters in animals. With advances in knowledge and technology, more PTXs are being identified, but little is known of their toxicology and the potential impact these toxins may have on public health in the long term. Without such information, adequate health-risk assessments for the consumption of shellfish contaminated with PTXs cannot be performed. This review gives a brief introduction to diarrhetic shellfish toxins, details the known toxicology and metabolism of PTXs in animals, and discusses known incidences of PTX poisoning in humans. D 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction to diarrhetic shellfish poisoning toxins Diarrhetic shellfish poisoning (DSP) is characterised by an acute gastrointestinal disturbance following the consumption of shellfish contaminated with either, or a combination of, okadaic acid (OA; Cohen et al., 1990; Draisci et al., 1998; Edebo et al., 1988), dinophysistoxins (DTX; James et al., 1999; Carmody et al., 1996), pectenotoxins (PTXs; Ishige et al., 1988; Yasumoto et al., 1985), yessotoxins (YTX; Yasumoto and Murata, 1993; Ogino et al., 1997), or azaspiracids (AZA) (Draisci et al., 2000; Ofuji et al., 1999). The classification of PTXs, YTXs and AZAs with the DSPs occurred due to the symptoms caused by them resembling those of the OA class, and the fact that these toxins can often be found in combination with known diarrhetic toxins in shellfish. None of these toxins have been confirmed to be diarrhoea causing and thus their classification is under review. Oral ingestion of these toxins can lead to the gastrointestinal disturbances of * Corresponding author. P.O. Box 594, Archerfield, QLD 4108, Australia. Tel.: +61-7-3247-9147, +61-408-551-658; fax: +61-7-3274-9003. E-mail address: [email protected] (V. Burgess). 1 P.O. Box 594, Archerfield, QLD 4108, Australia. Tel.: + 61-7-32479120; fax: + 61-7-3274-9003.
acute diarrhoea, nausea, vomiting, and abdominal pain symptoms can begin within 30 minutes of consumption of contaminated shellfish. No human mortalities to date have been reported from any cases of DSP poisoning, although there has been considerable morbidity resulting in hospitalisation. The DSPs are cyclic polyether compounds (Fig. 1) are found worldwide and pose a particular problem for the length of time the toxins remain active within the shellfish (Yasumoto et al., 1985). In addition, even with a sparse dinoflagellate population, where 200 cells/l or less are present, shellfish can still become sufficiently toxic to cause poisoning (Luckas, 1992). Thus, with the high morbidity and worldwide distribution, DSP presents a serious problem for shellfish industries and to public health. DSP toxins are produced by several of the Dinophysis species including D. acuta, D. fortii, D. acuminata, D. norvegica, D. mitra (Yasumoto and Murata, 1990; Lee et al., 1989), and D. caudata (Eaglesham et al., 2000), though they are also produced by benthic species such as Prorocentrum lima (Pillet and Houvenaghel, 1995; Pillet et al., 1995). DSP toxins have been detected in both axenic and nonaxenic batch cultures of Prorocentrum species thus indicating that production of toxins by the algae themselves is occurring and
0160-4120/01/$ – see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 1 ) 0 0 0 5 8 - 7
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Fig. 1. Structures of DSP toxins taken from James et al. (1999).
does not support the theory that a symbiont bacteria is exclusively producing the toxins (Kodama, 1988), as discussed later. This topic has been reviewed by Quilliam (1999). In various species of algae, an intraspecies variation and a seasonal variation in the toxin content and toxin profile is seen (Lee et al., 1989; Orr and Jones, 1998). Lee et al. (1989) postulated that these variances may account for the different toxin profiles seen in algae collected from different countries and noted that toxin production may be influenced by environmental factors rather than solely differences in genetic composition.
2. Regulation of algal toxins and public health Regulation of shellfish aquaculture is now in place to control outbreaks of poisonings (Hallegraeff et al., 1995). Different countries set different guidelines on the levels of various toxins permitted in their shellfish for domestic sale and for export. Control measures include chemical and biological procedures to identify and quantify the levels of toxins in shellfish before they are put on the market. The mouse bioassay, which is currently the most widely accepted test by the regulatory authorities, is now accompanied by analytical methods such as tandem mass spectrometry (MS-MS), or in vitro bioassays, which are more specific to
particular toxins. The IOC have released a manual entitled Design and implementation of some harmful algal monitoring systems gives good detail on alga bloom and toxin monitoring systems for Australia, New Zealand, and other parts of the world. In many countries, there are currently no set regulatory procedures or guidelines for the quantification and identification of DSP toxins in shellfish. At the time of compiling this review, Canada had no regulatory guidelines for DSP toxins, although they have adopted the 0.2-mg/g limit accepted in Japan (5 MU/100 g meat (Yasumoto and Murata, 1990). Some European countries have also adopted this limit as an informal guideline. Sweden has differing levels for their marketable shellfish, depending upon whether the shellfish is for export or domestic consumption. For example, Swedish regulators allow levels up to 60 mg OA equ/100 g mussel meat for shellfish heading for domestic consumption, but will only accept mussels with levels of 40 mg OA equ/100 g mussel meat for export, to ensure their exported mussels will be accepted by the buying countries (Hageltorn, 1988). Regulations for DSP recommend 16 – 20 mg ‘OA equivalents’ per 100 g shellfish meat (Garthwaite, 2000). Soames-Mraci (1995) stated, for 1995, that the maximum permitted levels in Australia for DSPs were 20– 60 mg OA/100 g shellfish meat, 2 mg OA/g hepatopancreas. However, it is not clear from this reference
V. Burgess, G. Shaw / Environment International 27 (2001) 275–283
whether these figures were enforced values or recommended guidelines, and where these figures were derived from.
3. General toxicology of diarrhetic shellfish poisonings Information on these toxins has appeared over the last decade giving evidence that some of these toxins are hepatotoxic and potent tumour promoters in laboratory animals (Takai et al., 1987; Arias et al., 1993; Fessard et al., 1994). The liver and intestine are target organs for these toxins due to the existence of specific uptake systems into these organs. This evidence may suggest that exposure to these toxins could have serious chronic implications for regular consumers of shellfish. Diarrhetic effects have only been sufficiently proven for OA and DTX-1 and DTX-3. The diarrhetic effects caused by OA were first investigated by Hamano et al. (1986). OA and DTX-1 are potent inhibitors of protein phosphatases (PP1 and PP2A) and this mode of action may be linked to the observed diarrhoea, degenerative changes in absorptive epithelium of the small intestine and to tumour promotion. Also, it has been suggested that OA may act by a similar mechanism to that caused by the bacteria Vibrio cholerae, which causes activation of adenylate cyclase (Cohen et al., 1990). This activation then affects a cascade of events of increased cAMP that activates cAMP-dependent protein kinase, which then phosphorylate certain proteins that regulate intestinal sodium secretion. Studies on YTXs have shown it not to cause diarrhoea or inhibit PP2A (Ogino et al., 1997). Thus, even though YTX is found in association with other DSPs in shellfish, the question has been raised as to whether it should be classified as a DSP. Many studies have investigated the effects of the DSPs on protein phosphatases and cellular regulation. Most of the studies have centered around OA. It was found that OA extracted from black sponges produces a dose-dependent inhibition of myosin phosphatase and enhances tension development (Takai et al., 1987). As no change was seen in the response caused by OA when neurotransmitters such as 5hydroxytryptamine, acetylcholine, and adrenaline were inhibited, Takai et al. (1987) went on to show that OA is a very potent inhibitor of PP1 and PP2A. These protein phosphatases dephosphorylate serine and threonine residues in the cytosol of mammalian cells. A group in Canada investigated additional six compounds isolated from diarrhetic shellfish and found many to be potent phosphatase inhibitors (Luu et al., 1993), but found PTX-1 and PTX-6 to be inactive against PP1 and PP2A. A good review of the protein phosphatase inhibition of OA is given by Cohen et al. (1990). This article also discusses how useful OA can be as a probe for the study of cellular regulation. Fujiki et al. (1988) discussed the tumour promoting capabilities of 25 chemical structures including several OA compounds isolated from marine organisms. The group demonstrated that OA induces ornithine decarboxylase in the glandular stomach of rats and
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suggested that OA may be a tumour promoter of rat stomach (Fujiki et al., 1988). Cordier et al. (2000) have conducted a study to investigate whether consumption of shellfish by humans contaminated with low levels of OA could influence rates of cancer in France, but their results were inconclusive.
4. Metabolism of algal toxins in marine animals Research has been conducted on the metabolism of these compounds within molluscs to determine how the shellfish avoid intoxication from feeding on the toxic algae and to establish which toxins are being consumed by humans when they eat the shellfish (Lassus et al., 1989; Pillet and Houvenaghel, 1995; Pillet et al., 1995; Suzuki et al., 2001). Studying the metabolism of the toxins within the shellfish may give us more of an idea of how these toxins are metabolised in humans. It was assumed that fish may have up-regulated metabolism systems to cope with the different algal toxins, but it has been shown that salmon are more sensitive to the toxins than rats (Fladmark et al., 1998). Studies on various toxins of the different algal syndromes in shellfish have shown significant differences in the toxin profile between the toxinproducing dinoflagellates and the contaminated bivalves. This indicates that metabolic transformation is occurring within the shellfish following consumption of the toxic algae (Oshima, 1995; Suzuki et al., 1998, 1999). Metabolising systems are known in shellfish that cause the conversion of algal toxins in vivo including glutathione and cysteine (Oshima, 1995) and zinc – hydrochloric reduction for PSP toxins (Shimizu, 1984). It was found that lethality is increased in marine animals that had never been previously exposed to certain algal toxins (Shumway and Cucci, 1987). This could indicate that there was an enzyme induction system involved in the detoxification or alteration of the toxin inside the molluscs for particular toxins, or the presence of another protective system such as a binding protein. New evidence has shown that ciguatoxic fish may contain a toxin-binding protein that may confer protection (Hahn et al., 1992). For ciguatoxins, acid-catalysed spiroisomerisation has been observed in the acid environment of the stomach of herbivorous fish (Lewis and Holmes, 1993) where incomplete biotransformation of the gambiertoxins to the ciguatoxins could be seen (Fig. 2). After accumulation in herbivores the toxins are transferred to carnivorous fish.
Fig. 2. Biotransformation of gambiertoxins (GTX) and ciguatoxins (CTX) (taken from Lewis and Holmes, 1993).
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Carnivorous fish have been shown to contain ciguatoxin and no gambiertoxin, indicating that any remaining gambiertoxin present in the herbivorous prey is completely biotransformed in the carnivorous fish (LeGrand et al., 1989; Murata et al., 1990; Lewis and Holmes, 1993). Some researchers have proposed that the potency of the different toxins can vary depending upon the stage in the growth cycle of the algae. Baden et al. (1989) suggested that the algae are in fact themselves subject to the toxicity and have their own metabolising enzymes to combat and detoxify the toxins throughout their different cycles. This was further investigated by Boland et al. (1993) who showed dinoflagellates could tolerate the high concentrations of the toxins they produce due to the upregulation of their protein phosphatase expression. However, this result questions the potential benefit of producing such toxins if the host producer is suffering from the effects of the poison itself. Thus, evidence of metabolism to protect the algae may support the hypothesis that symbiosis of the algae with a second toxin-producing bacterium or virus. An example of this is demonstrated with another group of algal toxins, where Kodama (1988) found that PSP toxins were produced by a bacterium of the Moraxella genus and caused the contamination of scallops with PSP toxins in the absence of any dinoflagellate algae in the laboratory. Other studies have shown variation between different species of shellfish and differing patterns of accumulation of certain toxins in preference to other toxins (Ishida et al., 1996). This observation of metabolism in the shellfish was also investigated by Lee et al. (1988) who found that DTX-3 was undetectable in D. fortii and suggested that acylation of DTX-1 to DTX-3 occurred in the hepatopancreas of scallops. It may be possible that by studying the metabolism of the toxins within the shellfish that food poisoning could prevented by altering the metabolism of the toxin in the shellfish either in aquaculture beds or during cooking. Such research indicates that, from a public health aspect of shellfish poisoning, it would be more appropriate to carry out studies using toxins extracted and purified from marine animals rather than from algal cultures.
evidence (Edebo et al., 1988; Terao et al., 1986). PTX-2 was first identified in Europe in the Northern Adriatic Sea but it was then known to be more abundant in Japan (Draisci et al., 1996). Studies on the chemistry of PTXs have facilitated the elucidation of new structures and there has been rapid progression in the identification of new analogues. It is believed that several of the PTXs are derived from a parent PTX, where the parent molecule is metabolised within the scallops to form other PTX analogues (Yasumoto and Murata, 1990).
6. Structural variation of the pectenotoxins and their metabolism in shellfish Many methodologies have been employed for the extraction of PTXs from algal bloom samples and from shellfish. Ideally, for the study of PTXs it would be convenient to culture the causative algae and harvest the toxins from dense algal populations, but the Dinophysis species are notoriously difficult to culture (Hallegraeff et al., 1995). Thus, most extractions are performed on the whole shellfish, their gut contents, or algal samples collected during a red tide. Because of this, the clean-up process and purification of PTXs can be laborious, costly, and produce a poor yield of toxin. Analytical difficulties can arise with the investigation of the PTX due to their instability under certain environmental conditions. For example, Sasaki et al. (1998) identified PTXs isolated from the digestive glands of the scallop Patinopecten yessoensis named PTX-4 and PTX-7. At the time of their article, eight PTXs (named PTX-1-7, and PTX-10) had been recognised from this scallop and Sasaki et al. (1998) described two new structures, PTX-8 and PTX-9, which were produced following chemical treatment of another PTX. Sasaki et al. (1998) demonstrated that, upon standing in aqueous acetonitrile, PTX-7 gradually transformed to PTX-6 indicating acid-catalysed interconversion occurred around the carboxyl group. Addition of trifluroacetic acid (0.1% v/v) accelerated the conversion and also caused the formation of another isomeric PTX (PTX-9). Despite the absence of a carboxyl group in PTX-4, under the same acid conditions, PTX-4 and PTX-1 produced a new compound PTX-8. Yasumoto
5. Pectenotoxins PTXs are a group of toxins associated with DSP and isolated from algae commonly known to produce other DSPs such as OA and DTX-1. Examples of such algae include the dinoflagellates D. acuta, D. fortii, D. acuminata and D. caudata. Although classified with the DSPs, as the PTXs are often found in combination with other DSPs in shellfish, there is debate over whether these toxins should be classified as DSP toxins. Some groups have found mild diarrhetic effects caused by the administration of PTXs (Ishige et al., 1988) while others have found no such
Table 1 Some physiochemical parameters for the PTXs (compiled from the article by Yasumoto et al., 1985) PTX
lmax (nm)
Rf in TLC
MS m/z
1 2 3 4 5
235 235 235 235 235
0.43 0.71 0.49 0.53 0.41
875 859 875 875 877
Minimum lethal dose (mg/kg, ip) 250 260
V. Burgess, G. Shaw / Environment International 27 (2001) 275–283
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Fig. 3. The PTXs (taken from Sasaki et al., 1998).
et al. (1985) carried out some early structural determination studies on the PTXs (Table 1). Later studies by Sasaki et al. (1998) found the structural variation between the PTXs to be determined by the nature of the substituent at C-18 (Fig. 3). Heating of the PTXs, such as would occur during cooking of shellfish, causes epimerisation of PTX-2-SA in the same manner as occurs when PTX-2-SA is left dry in a glass container (Eaglesham et al., 2000). Sasaki et al. (1998) also proposed that the PTX-1, PTX3, and PTX-6 are derived from PTX-2 (Fig. 3). Other congeners have not yet had their structures determined and investigation of their origin is not possible. The results from the investigations of Sasaki et al. (1998) indicated that PTX-6 and PTX-7 are isomeric structures as are PTX-1 and PTX-4. Both sets shared a common carbon skeleton with differing substituents at the C-18. Thus, a conclusion was reached that PTX-4 and PTX-7 are stereoisomers of 1 and 6, respectively. Lee et al. (1988), while identifying PTX production in D. fortii and PTX detection in scallops, found only PTX-2 in the algae and suggested that oxidation of the 43-methyl group of PTX-2 takes place in the hepatopancreas of the scallop. Studies on the metabolism of PTXs within scallops by Suzuki et al. (1998) indicated that PTX-2 is converted via an oxidation step on the 43-methyl group to PTX-6. This, too, was supported by the fact that PTX-2 was detected in both extracts of D. fortii and in extracts of scallop gut, whereas PTX-6 could only be detected within the gut of the scallop and not in the algal extracts. Another recently identified analogue is pectenotoxin-2 seco acid (PTX-2-SA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2-SA) isolated from greenshell mussels (Daiguji et al., 1998) (Fig. 1). It is characteristic of the PTXs to have a large lactone ring as seen in Fig. 1B, but, unlike these structures, the seco acids have an open chain structure (Fig. 1C). Daiguji et al. (1998) did not find these seco acids
to be cytotoxic towards KB cells at a dose of 1.8 mg/ml, while PTX-2 at a dose of 0.05 mg/ml was cytotoxic. This indicates that the cyclic structure of PTX-2 is required for toxicity. Thus, the conversion of PTX-2 to the seco acid may be protective for detoxification in the shellfish. Suzuki et al. (2001) recently compared the toxin profiles of the seco acids from D. acuta to that of the greenshell and blue mussels isolated from an area when D. acuta was blooming in New Zealand. They found the major PTX toxin in the algae to be PTX-2, whereas both of the shellfish species contained PTX-2-SA and 7-epi-PTX-2-SA (Table 2).
7. Toxicology of the pectenotoxins in mammals The PTXs are hepatotoxic, tumour promoters, and cause apoptosis in rat and salmon hepatocytes (Fladmark et al.,
Table 2 Percentage composition of the different toxins in D. acuta, greenshell mussels, and blue mussels for samples taken in 1998 and in 1996 (taken from Suzuki et al., 2001) Samples (Year of collection) D. acuta (1998) Greenshell mussels (1998) Blue mussels Greenshell mussels (1996) Blue mussels (1996)
Profile (%) PTX-2
PTX-2-SA
7-epi-PTX-2-SA
Other PTXs
92.2 3.1
7.8 86.6
– 10.4
– –
3.8 1.6
88.2 56.6
8.0 41.8
– –
0.9
57.9
41.2
–
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1998). When rat and salmon hepatocytes were dosed with 2 mM PTX-1, apoptotic effects showing extreme nuclear and cellular shrinkage and chromatin hypercondensation were observed within 1 h. The salmon hepatocytes appear more sensitive to the PTXs as demonstrated by the pronounced cell shrinkage that was observed. Cell death was found to be caused by apoptosis rather than by necrosis. This was determined by the observation of a lack of trypan blue uptake and the chromatin condensation typical of apoptosis. In addition, the caspase inhibitor ZVAD-fmk was shown to inhibit the action of PTX-1, which also supports that apoptosis was the cause of cell death. PTX-2 has been found to be potently cytotoxic to the human lung (A-549), colon (HT29), and breast (MCF-7) cancer cell lines, but also cytotoxic to several other cell lines including ovarian, renal, lung, CNS, melanoma, and breast cancer cell lines (Jung et al., 1995). From studies with the DNA cleavage assay and the rat plasma membrane assay, Jung et al. (1995) found that PTX-2 neither exerts its cytotoxic activity by blocking DNA synthesis nor by blocking reduction –oxidation processes within the cell membrane. Pathologically, PTX-1 has been shown to be hepatotoxic causing rapid necrosis of hepatocytes, in particular to periportal regions of hepatic lobules (Terao et al., 1986). Further, PTX was not a causative agent of the diarrhetic symptoms of DSP with dosing levels of 1000, 700, 500, 250, and 150 mg/kg via i.p. (Terao et al., 1986). Mice treated with an intraperitoneal dose of 1000 mg/kg of PTX showed severe congestion of their livers, and the surface of the livers appeared finely granulated within 60 min of the intraperitoneal injection of PTX-1. No pathological changes in the large and small intestines or in any other visceral organs were observable. Multiple large vacuoles appeared around the periportal regions of the lobules. The vacuoles did not stain with neutral red, which indicates changes in neutral lipids, and several of the vacuoles located in the subcapsular region contained erythrocytes. Hyaline droplets were seen in the hepatocytes that developed vacuoles. Electron microscopic observation showed flattening of the microvilli on the hepatocytes in the periportal region and also showed the plasma membrane to be invaginated into its cytoplasm.
These observations in the liver were similar to those observed by Aune (1988) who dosed freshly isolated hepatocytes with various DSPs. More recently, Zhou et al. (1994) used primary cultured liver cells from chick embryos to study the effects of PTX-1 on the cytoskeletons of liver cells with concentrations ranging from 5 ng/ml to 50 mg/ml for 0.5 –24 h. Antitubulin staining revealed that the perinuclear regions had become sparse in microtubules and the remaining perinuclear regions appeared disorganised, while actin microfilaments were also disrupted. Zhou et al. (1994) also found that, for doses up to 5 ng/ml or less, removal of the toxin within 4 h from the culture media revealed that the morphology induced by PTX-1 was reversible. The study by Terao et al. (1986) found that PTX-1 caused damage to the hepatocytes in the periportal area of the lobule. The periportal area approximates to zone 1 of the lobule, which is known to receive blood from the afferent venules and so is rich in oxygen and nutrients (Turton and Hooson, 1998; Timbrell, 1996). Because of this, the hepatocytes in the periportal lobule are more equipped for boxidation of fats and there are also increased levels of glutathione and glutathione reductase in the hepatocytes of this zone. Thus, toxicity seen in this area may indicate that the PTXs are metabolised and removed from the body by a simple conjugation reaction with for example, gluathione or glucuronidation of the hydroxyl groups rather than through a P450 reaction, which is generally located in the zone 3 region and centrilobular damage would be expected if P450 metabolism was the primary metabolic reaction. This finding has been elaborated in a study by Yoon and Kim (1997) where mice were dosed with PTX-2 and a decrease in hepatic microsomal protein content was observed. However, the total P450 content, cytochrome b5, NADPHcytochrome c reductase, and aminopyrine N-demethylase activities were not affected (Yoon and Kim, 1997). Glutathione content was also unaltered, but elevated serum enzyme levels of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase were found (Yoon and Kim, 1997). Changes in these enzymes are known to indicate that hepatocyte damage has occurred (Turton and Hooson, 1998).
Table 3 Summary of toxicity concentrations for various DSP algal toxins in mice Toxin class DSP OA DTX-1 DTX-3 PTX-1 PTX-II PTX-11 PTX1-6 YTX
LD50 toxicity
Reference
Kg Kg Kg Kg Kg Kg Kg
1
100 mg Kg > 1000 mg Kg
1
192 200 166 500 250 260 250
mg mg mg mg mg mg mg
1 1 1 1 1 1
1
i.v. i.v. i.v. i.p. i.p. i.p. i.p.
Tachibana et al., 1981 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Jung et al., 1995
i.p. p.o
Ogino, 1997
LD99 toxicity 200 mg Kg 160 mg Kg 500 mg Kg
1 1 1
Reference i.p. i.p. i.p.
160 – 170 mg Kg 1 i.p. 100 mg Kg 1 i.p.
Yasumoto and Murata, 1990 Yasumoto and Murata, 1990 Yasumoto and Murata, 1990
Yasumoto and Murata, 1990 Yasumoto and Murata, 1990
V. Burgess, G. Shaw / Environment International 27 (2001) 275–283
Ishige et al. (1988) conducted an oral toxicity study with PTX-2. At doses ranging from 250 to 2000 mg/kg, PTX-2 caused diarrhetic symptoms accompanied by severe mucosal injuries in the small intestine between 30 and 360 min after dosing by oral injection. Hepatic injuries were also observed as hyaline droplet degeneration in hepatocytes around peripheral zones of the hepatic lobules. Spongiosis lesions of hydropic vacuolar degeneration could be seen in the intermediate zone. Dilatations of the sinusoids and hydropic swelling of the endothelial cells were also observed. The study conducted by Ishige et al. (1988) was one of few oral studies conducted with the PTXs. Levels used in the Ishige et al. (1988) study were very high when compared to what we know of the human situation of shellfish poisoning. This may indicate that humans could be more sensitive to PTX toxins than mice. If this is the case, absorption and metabolism studies must be conducted in the mice to understand the mechanisms of toxicity for the PTXs. Very few studies have been done in mammalian systems that could be directly related to the human situation of shellfish poisoning with regard to the metabolism of the PTXs. With reference to Table 3, it can be seen that, in mice, the LD50 toxicity of PTX-1 and PTX-2 has been determined to be 250 mg/kg, ip, for PTX-1 and 260 mg/kg, ip, for PTX-2. These figures are higher than the LD50 value for DTX-3 (500 mg/kg) but lower toxicity than that seen for DTX-1 (166 mg/kg) or OA at 200 mg/kg. The highest toxicity for DSP toxins is seen with the YTXs with an i.p. LD50 of 100 mg/kg. By the oral route, yessotoxin was not found to be toxic at 1000 mg/kg. It has been found that the oral toxicity of OA is 25– 50 times lower than that seen from i.p. dosing (Aune et al., 1998). A review article by Yasumoto and Murata (1990) stated that the LD99 in mice (intraperitoneal) for PTX-1 –6 ranged from 106 to 770 mg/kg. This study did not define which PTX isomer caused the greatest toxicity, and did not provide detail of any of the pathologies observed. LD50 values are useful in providing information for regulatory and safety assessments and indicating the potency of the toxin but provide no information on the mechanisms of toxicity and other pathological processes that would aid in the development of antidotes or treatments for the toxins.
8. Pectenotoxins and public health All the in vivo studies with the PTXs discussed above have used very high dosing levels compared with the doses known to cause illness in the human situation. PTXs have been associated with severe diarrhetic illness resulting in hospitalisation. One such incident occurred during December of 1997 when over 100 people living in the Eastern states of Australia were poisoned following consumption of pipis harvested off the New South Wales (NSW) coastline. This particular incident included 56 cases of hospitalisation in NSW alone. Symptoms included nausea, vomiting, and
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diarrhoea. The shellfish responsible for the poisoning were found to contain PTX-2-SA and low levels of PTX-2 (Eaglesham et al., 2000). Another poisoning incident involving the consumption of pipis (Donax delatoides) occurred on North Stradbroke Island (QLD, Australia) in March 2000 where an elderly woman became seriously ill after eating pipis from one of the local beaches. To investigate this incident, fresh pipis were collected from a pristine beach on the island with no sewage outlets. High levels of PTX-2-SA were found in the shellfish that caused the illness (Eaglesham et al., 2000). Pipis can metabolise PTX-2 to PTX-2-SA which explains the different toxin profiles seen between the shellfish and the phytoplankton at the time of the Stradbroke poisoning. Thus, it seems possible that these seco acids are responsible for the diarrhetic symptoms seen or that once ingested, they may convert back to PTX-2 prior to absorption in to the body perhaps in the strongly acid or alkaline environment of the digestive tract. Alternatively, the toxin may be absorbed as the seco acid and metabolised in the liver. No toxicity data has been published on the PTX-2 seco acid isomers to date but toxicopathologies are currently under investigation (Burgess et al., 2001). By analysis of the pipis from the poisoning incident, the amount of toxin consumed by the patient was estimated. It was found that the pipis contained approximately 300 mg/kg of pipis. The average meal consists of approximately 0.5 kg of pipis. Therefore, the person consumed approximately 150 mg of PTX-2-SA during the meal. For an average 70-kg person, this equates to a dose of 2 mg PTX-2-SA/kg body weight leading to illness in humans. This figure is considerably lower than the doses used in the toxicity studies in mice described in this review. The amount of DTX-1 sufficient to induce gastroenteritis in a human is 32 mg when eaten (Yasumoto et al., 1985). This also, is a very low figure compared to the doses used in the toxicology studies for DTXs described in this article. In light of this information, the question should be posed as to why the oral toxicity studies are conducted with such high doses in mice? In addition, the question is raised ‘‘are humans more sensitive to DSP toxins than mice?’’ The symptoms experienced at lower doses may relate to differences in metabolism for the DSPs or other physiological processes such as absorption mechanisms and simply to differences between intestinal surface area to body weight ratios. It is fundamental to our understanding of the process of poisoning to gather information on the mechanisms of toxicity of these toxins. It is not possible to conduct an accurate health-risk assessment without such information.
9. Conclusions The PTXs are known to cause severe acute illness in humans. Research has shown that these toxins are potently cytotoxic, tumour promoters, and cause necrosis to hepato-
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cytes. Nothing is known of the chronic toxicology of PTXs or the potential implications to public health in the long term. This information is needed to perform health-risk assessments for consumption of contaminated shellfish, and for guidance in the regulation of permitted toxin levels in shellfish that are sold for human consumption. It is evident, for an appropriate health-risk assessment to be performed, more toxicology and metabolism work is required on the PTXs, especially where the oral toxicity is concerned. Research must be conducted with relevance to the human situation of shellfish poisoning. It is also important that research is undertaken to assess the effects of consuming several of the DSP toxins in one meal, as often occurs in the real-life situation where shellfish can be contaminated with a combination of DSP toxins.
Acknowledgments NRCET is funded by the National Health and Medical Research Council, Queensland Health, Griffith University, Queensland University of Technology and the University of Queensland.
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Shumway SE, Cucci L. The effects of toxic dinoflagellate Protogonyaulax tamarensis on feeding and behaviour of bivalve molluscs. Aquat Toxicol 1987;10:9 – 27. Soames-Mraci CP. Shellfish poisoning: public health risks, quality assurance and analytical detection. Chem Aust. 1995;22 – 5. Suzuki T, Mitsuya T, Matsubara H, Yamasaki M. Determination of pectenotoxin-2 after solid-phase extraction from seawater and from the dinoflagellate Dinophysis fortii by liquid chromatography with electrospray mass spectrometry and ultraviolet detection. Evidence of oxidation of pectenotoxin-2 to pectenotoxin-6 in scallops. J Chromatogr A 1998; 815:155 – 60. Suzuki T, Ota H, Yamasaki M. Direct evidence of transformation of dinophysistoxin-1 to 7-O-acyl-dinophysistoxin-1 (dinophysistoxin-3) in the scallop Patinopecten yessoensis. Toxicon 1999;37(7):187 – 98. Suzuki T, MacKenzie L, Stirling D, Adamson J. Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the new Zealand greenshell mussel Perna canaliculus. Toxicon 2001;39(4):507 – 14. Tachibana K, Schuer PJ, Tsukitani Y, Kikuchi H, Engen DV, Clardy J, Gopichaud Y, Schmitz FJ. Okadaic acid, a cytotoxic polyether from tow marine sponges of the genus halichondoria. J Am Chem Soc 1981;103:2469 – 71. Takai A, Bialojan C, Troschka M, Ruegg JR. Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin. FEBS 1987;217(1):81 – 4. Terao K, Ito E, Yanagi T, Yasumoto T. Histopathological studies on experimental marine toxin poisoning: I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 1986;24(11 – 12):1141 – 51. Timbrell JA. Principles of biochemical toxicology. 2nd ed. London: Taylor & Francis, 1996. Turton J, Hooson J, editors. Target organ pathology, a basic text. London: Taylor & Francis, 1998. Yasumoto T, Murata M. Polyether toxins involved in seafood poisoning. ACS Symp Ser 1990;418:120 – 32. Yasumoto T, Murata M. Marine toxins. Chem Rev 1993;93:1897 – 909. Yasumoto T, Murata M, Oshima Y, Sano M. Diarrhetic shellfish poisons. Tetrahedron 1985;41(6):1019 – 25. Yoon MY, Kim YC. Acute toxicity of pectenotoxin-2 and its effects on hepatic-metabolising enzyme system in mice. Korean J Toxicol 1997; 13(3):183 – 6. Zhou Z-h, Masatoshi K, Terao K, Shimada Y. Effects of pectenotoxin-1 on liver cells in vitro. Nat Toxins 1994;2:132 – 5.
Pectenotoxins — an issue for public health A review of their comparative toxicology and metabolism Vanessa Burgessa,b,*, Glen Shawb,1 a
National Research Centre for Environmental Toxicology (NRCET), 39 Kessels Road, Coopers Plains, Brisbane, QLD 4108, Australia b School of Public Health, Griffith University, Logan Campus, Brisbane, QLD 4111, Australia Received 8 December 2000; accepted 18 May 2001
Abstract Pectenotoxins (PTXs) are a group of toxins associated with diarrhetic shellfish poisoning (DSP) and isolated from DSP toxin-producing dinoflagellate algae. Consumption of shellfish contaminated with PTXs has been associated with incidences of severe diarrhetic illness resulting in hospitalisation. Concern has been raised for public health following the discovery that these toxins are not only hepatotoxic and can cause diarrhetic effects in mammals, but that they are potently cytotoxic to human cancer cell lines and have been found to be tumour promoters in animals. With advances in knowledge and technology, more PTXs are being identified, but little is known of their toxicology and the potential impact these toxins may have on public health in the long term. Without such information, adequate health-risk assessments for the consumption of shellfish contaminated with PTXs cannot be performed. This review gives a brief introduction to diarrhetic shellfish toxins, details the known toxicology and metabolism of PTXs in animals, and discusses known incidences of PTX poisoning in humans. D 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction to diarrhetic shellfish poisoning toxins Diarrhetic shellfish poisoning (DSP) is characterised by an acute gastrointestinal disturbance following the consumption of shellfish contaminated with either, or a combination of, okadaic acid (OA; Cohen et al., 1990; Draisci et al., 1998; Edebo et al., 1988), dinophysistoxins (DTX; James et al., 1999; Carmody et al., 1996), pectenotoxins (PTXs; Ishige et al., 1988; Yasumoto et al., 1985), yessotoxins (YTX; Yasumoto and Murata, 1993; Ogino et al., 1997), or azaspiracids (AZA) (Draisci et al., 2000; Ofuji et al., 1999). The classification of PTXs, YTXs and AZAs with the DSPs occurred due to the symptoms caused by them resembling those of the OA class, and the fact that these toxins can often be found in combination with known diarrhetic toxins in shellfish. None of these toxins have been confirmed to be diarrhoea causing and thus their classification is under review. Oral ingestion of these toxins can lead to the gastrointestinal disturbances of * Corresponding author. P.O. Box 594, Archerfield, QLD 4108, Australia. Tel.: +61-7-3247-9147, +61-408-551-658; fax: +61-7-3274-9003. E-mail address: [email protected] (V. Burgess). 1 P.O. Box 594, Archerfield, QLD 4108, Australia. Tel.: + 61-7-32479120; fax: + 61-7-3274-9003.
acute diarrhoea, nausea, vomiting, and abdominal pain symptoms can begin within 30 minutes of consumption of contaminated shellfish. No human mortalities to date have been reported from any cases of DSP poisoning, although there has been considerable morbidity resulting in hospitalisation. The DSPs are cyclic polyether compounds (Fig. 1) are found worldwide and pose a particular problem for the length of time the toxins remain active within the shellfish (Yasumoto et al., 1985). In addition, even with a sparse dinoflagellate population, where 200 cells/l or less are present, shellfish can still become sufficiently toxic to cause poisoning (Luckas, 1992). Thus, with the high morbidity and worldwide distribution, DSP presents a serious problem for shellfish industries and to public health. DSP toxins are produced by several of the Dinophysis species including D. acuta, D. fortii, D. acuminata, D. norvegica, D. mitra (Yasumoto and Murata, 1990; Lee et al., 1989), and D. caudata (Eaglesham et al., 2000), though they are also produced by benthic species such as Prorocentrum lima (Pillet and Houvenaghel, 1995; Pillet et al., 1995). DSP toxins have been detected in both axenic and nonaxenic batch cultures of Prorocentrum species thus indicating that production of toxins by the algae themselves is occurring and
0160-4120/01/$ – see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 1 ) 0 0 0 5 8 - 7
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Fig. 1. Structures of DSP toxins taken from James et al. (1999).
does not support the theory that a symbiont bacteria is exclusively producing the toxins (Kodama, 1988), as discussed later. This topic has been reviewed by Quilliam (1999). In various species of algae, an intraspecies variation and a seasonal variation in the toxin content and toxin profile is seen (Lee et al., 1989; Orr and Jones, 1998). Lee et al. (1989) postulated that these variances may account for the different toxin profiles seen in algae collected from different countries and noted that toxin production may be influenced by environmental factors rather than solely differences in genetic composition.
2. Regulation of algal toxins and public health Regulation of shellfish aquaculture is now in place to control outbreaks of poisonings (Hallegraeff et al., 1995). Different countries set different guidelines on the levels of various toxins permitted in their shellfish for domestic sale and for export. Control measures include chemical and biological procedures to identify and quantify the levels of toxins in shellfish before they are put on the market. The mouse bioassay, which is currently the most widely accepted test by the regulatory authorities, is now accompanied by analytical methods such as tandem mass spectrometry (MS-MS), or in vitro bioassays, which are more specific to
particular toxins. The IOC have released a manual entitled Design and implementation of some harmful algal monitoring systems gives good detail on alga bloom and toxin monitoring systems for Australia, New Zealand, and other parts of the world. In many countries, there are currently no set regulatory procedures or guidelines for the quantification and identification of DSP toxins in shellfish. At the time of compiling this review, Canada had no regulatory guidelines for DSP toxins, although they have adopted the 0.2-mg/g limit accepted in Japan (5 MU/100 g meat (Yasumoto and Murata, 1990). Some European countries have also adopted this limit as an informal guideline. Sweden has differing levels for their marketable shellfish, depending upon whether the shellfish is for export or domestic consumption. For example, Swedish regulators allow levels up to 60 mg OA equ/100 g mussel meat for shellfish heading for domestic consumption, but will only accept mussels with levels of 40 mg OA equ/100 g mussel meat for export, to ensure their exported mussels will be accepted by the buying countries (Hageltorn, 1988). Regulations for DSP recommend 16 – 20 mg ‘OA equivalents’ per 100 g shellfish meat (Garthwaite, 2000). Soames-Mraci (1995) stated, for 1995, that the maximum permitted levels in Australia for DSPs were 20– 60 mg OA/100 g shellfish meat, 2 mg OA/g hepatopancreas. However, it is not clear from this reference
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whether these figures were enforced values or recommended guidelines, and where these figures were derived from.
3. General toxicology of diarrhetic shellfish poisonings Information on these toxins has appeared over the last decade giving evidence that some of these toxins are hepatotoxic and potent tumour promoters in laboratory animals (Takai et al., 1987; Arias et al., 1993; Fessard et al., 1994). The liver and intestine are target organs for these toxins due to the existence of specific uptake systems into these organs. This evidence may suggest that exposure to these toxins could have serious chronic implications for regular consumers of shellfish. Diarrhetic effects have only been sufficiently proven for OA and DTX-1 and DTX-3. The diarrhetic effects caused by OA were first investigated by Hamano et al. (1986). OA and DTX-1 are potent inhibitors of protein phosphatases (PP1 and PP2A) and this mode of action may be linked to the observed diarrhoea, degenerative changes in absorptive epithelium of the small intestine and to tumour promotion. Also, it has been suggested that OA may act by a similar mechanism to that caused by the bacteria Vibrio cholerae, which causes activation of adenylate cyclase (Cohen et al., 1990). This activation then affects a cascade of events of increased cAMP that activates cAMP-dependent protein kinase, which then phosphorylate certain proteins that regulate intestinal sodium secretion. Studies on YTXs have shown it not to cause diarrhoea or inhibit PP2A (Ogino et al., 1997). Thus, even though YTX is found in association with other DSPs in shellfish, the question has been raised as to whether it should be classified as a DSP. Many studies have investigated the effects of the DSPs on protein phosphatases and cellular regulation. Most of the studies have centered around OA. It was found that OA extracted from black sponges produces a dose-dependent inhibition of myosin phosphatase and enhances tension development (Takai et al., 1987). As no change was seen in the response caused by OA when neurotransmitters such as 5hydroxytryptamine, acetylcholine, and adrenaline were inhibited, Takai et al. (1987) went on to show that OA is a very potent inhibitor of PP1 and PP2A. These protein phosphatases dephosphorylate serine and threonine residues in the cytosol of mammalian cells. A group in Canada investigated additional six compounds isolated from diarrhetic shellfish and found many to be potent phosphatase inhibitors (Luu et al., 1993), but found PTX-1 and PTX-6 to be inactive against PP1 and PP2A. A good review of the protein phosphatase inhibition of OA is given by Cohen et al. (1990). This article also discusses how useful OA can be as a probe for the study of cellular regulation. Fujiki et al. (1988) discussed the tumour promoting capabilities of 25 chemical structures including several OA compounds isolated from marine organisms. The group demonstrated that OA induces ornithine decarboxylase in the glandular stomach of rats and
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suggested that OA may be a tumour promoter of rat stomach (Fujiki et al., 1988). Cordier et al. (2000) have conducted a study to investigate whether consumption of shellfish by humans contaminated with low levels of OA could influence rates of cancer in France, but their results were inconclusive.
4. Metabolism of algal toxins in marine animals Research has been conducted on the metabolism of these compounds within molluscs to determine how the shellfish avoid intoxication from feeding on the toxic algae and to establish which toxins are being consumed by humans when they eat the shellfish (Lassus et al., 1989; Pillet and Houvenaghel, 1995; Pillet et al., 1995; Suzuki et al., 2001). Studying the metabolism of the toxins within the shellfish may give us more of an idea of how these toxins are metabolised in humans. It was assumed that fish may have up-regulated metabolism systems to cope with the different algal toxins, but it has been shown that salmon are more sensitive to the toxins than rats (Fladmark et al., 1998). Studies on various toxins of the different algal syndromes in shellfish have shown significant differences in the toxin profile between the toxinproducing dinoflagellates and the contaminated bivalves. This indicates that metabolic transformation is occurring within the shellfish following consumption of the toxic algae (Oshima, 1995; Suzuki et al., 1998, 1999). Metabolising systems are known in shellfish that cause the conversion of algal toxins in vivo including glutathione and cysteine (Oshima, 1995) and zinc – hydrochloric reduction for PSP toxins (Shimizu, 1984). It was found that lethality is increased in marine animals that had never been previously exposed to certain algal toxins (Shumway and Cucci, 1987). This could indicate that there was an enzyme induction system involved in the detoxification or alteration of the toxin inside the molluscs for particular toxins, or the presence of another protective system such as a binding protein. New evidence has shown that ciguatoxic fish may contain a toxin-binding protein that may confer protection (Hahn et al., 1992). For ciguatoxins, acid-catalysed spiroisomerisation has been observed in the acid environment of the stomach of herbivorous fish (Lewis and Holmes, 1993) where incomplete biotransformation of the gambiertoxins to the ciguatoxins could be seen (Fig. 2). After accumulation in herbivores the toxins are transferred to carnivorous fish.
Fig. 2. Biotransformation of gambiertoxins (GTX) and ciguatoxins (CTX) (taken from Lewis and Holmes, 1993).
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Carnivorous fish have been shown to contain ciguatoxin and no gambiertoxin, indicating that any remaining gambiertoxin present in the herbivorous prey is completely biotransformed in the carnivorous fish (LeGrand et al., 1989; Murata et al., 1990; Lewis and Holmes, 1993). Some researchers have proposed that the potency of the different toxins can vary depending upon the stage in the growth cycle of the algae. Baden et al. (1989) suggested that the algae are in fact themselves subject to the toxicity and have their own metabolising enzymes to combat and detoxify the toxins throughout their different cycles. This was further investigated by Boland et al. (1993) who showed dinoflagellates could tolerate the high concentrations of the toxins they produce due to the upregulation of their protein phosphatase expression. However, this result questions the potential benefit of producing such toxins if the host producer is suffering from the effects of the poison itself. Thus, evidence of metabolism to protect the algae may support the hypothesis that symbiosis of the algae with a second toxin-producing bacterium or virus. An example of this is demonstrated with another group of algal toxins, where Kodama (1988) found that PSP toxins were produced by a bacterium of the Moraxella genus and caused the contamination of scallops with PSP toxins in the absence of any dinoflagellate algae in the laboratory. Other studies have shown variation between different species of shellfish and differing patterns of accumulation of certain toxins in preference to other toxins (Ishida et al., 1996). This observation of metabolism in the shellfish was also investigated by Lee et al. (1988) who found that DTX-3 was undetectable in D. fortii and suggested that acylation of DTX-1 to DTX-3 occurred in the hepatopancreas of scallops. It may be possible that by studying the metabolism of the toxins within the shellfish that food poisoning could prevented by altering the metabolism of the toxin in the shellfish either in aquaculture beds or during cooking. Such research indicates that, from a public health aspect of shellfish poisoning, it would be more appropriate to carry out studies using toxins extracted and purified from marine animals rather than from algal cultures.
evidence (Edebo et al., 1988; Terao et al., 1986). PTX-2 was first identified in Europe in the Northern Adriatic Sea but it was then known to be more abundant in Japan (Draisci et al., 1996). Studies on the chemistry of PTXs have facilitated the elucidation of new structures and there has been rapid progression in the identification of new analogues. It is believed that several of the PTXs are derived from a parent PTX, where the parent molecule is metabolised within the scallops to form other PTX analogues (Yasumoto and Murata, 1990).
6. Structural variation of the pectenotoxins and their metabolism in shellfish Many methodologies have been employed for the extraction of PTXs from algal bloom samples and from shellfish. Ideally, for the study of PTXs it would be convenient to culture the causative algae and harvest the toxins from dense algal populations, but the Dinophysis species are notoriously difficult to culture (Hallegraeff et al., 1995). Thus, most extractions are performed on the whole shellfish, their gut contents, or algal samples collected during a red tide. Because of this, the clean-up process and purification of PTXs can be laborious, costly, and produce a poor yield of toxin. Analytical difficulties can arise with the investigation of the PTX due to their instability under certain environmental conditions. For example, Sasaki et al. (1998) identified PTXs isolated from the digestive glands of the scallop Patinopecten yessoensis named PTX-4 and PTX-7. At the time of their article, eight PTXs (named PTX-1-7, and PTX-10) had been recognised from this scallop and Sasaki et al. (1998) described two new structures, PTX-8 and PTX-9, which were produced following chemical treatment of another PTX. Sasaki et al. (1998) demonstrated that, upon standing in aqueous acetonitrile, PTX-7 gradually transformed to PTX-6 indicating acid-catalysed interconversion occurred around the carboxyl group. Addition of trifluroacetic acid (0.1% v/v) accelerated the conversion and also caused the formation of another isomeric PTX (PTX-9). Despite the absence of a carboxyl group in PTX-4, under the same acid conditions, PTX-4 and PTX-1 produced a new compound PTX-8. Yasumoto
5. Pectenotoxins PTXs are a group of toxins associated with DSP and isolated from algae commonly known to produce other DSPs such as OA and DTX-1. Examples of such algae include the dinoflagellates D. acuta, D. fortii, D. acuminata and D. caudata. Although classified with the DSPs, as the PTXs are often found in combination with other DSPs in shellfish, there is debate over whether these toxins should be classified as DSP toxins. Some groups have found mild diarrhetic effects caused by the administration of PTXs (Ishige et al., 1988) while others have found no such
Table 1 Some physiochemical parameters for the PTXs (compiled from the article by Yasumoto et al., 1985) PTX
lmax (nm)
Rf in TLC
MS m/z
1 2 3 4 5
235 235 235 235 235
0.43 0.71 0.49 0.53 0.41
875 859 875 875 877
Minimum lethal dose (mg/kg, ip) 250 260
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Fig. 3. The PTXs (taken from Sasaki et al., 1998).
et al. (1985) carried out some early structural determination studies on the PTXs (Table 1). Later studies by Sasaki et al. (1998) found the structural variation between the PTXs to be determined by the nature of the substituent at C-18 (Fig. 3). Heating of the PTXs, such as would occur during cooking of shellfish, causes epimerisation of PTX-2-SA in the same manner as occurs when PTX-2-SA is left dry in a glass container (Eaglesham et al., 2000). Sasaki et al. (1998) also proposed that the PTX-1, PTX3, and PTX-6 are derived from PTX-2 (Fig. 3). Other congeners have not yet had their structures determined and investigation of their origin is not possible. The results from the investigations of Sasaki et al. (1998) indicated that PTX-6 and PTX-7 are isomeric structures as are PTX-1 and PTX-4. Both sets shared a common carbon skeleton with differing substituents at the C-18. Thus, a conclusion was reached that PTX-4 and PTX-7 are stereoisomers of 1 and 6, respectively. Lee et al. (1988), while identifying PTX production in D. fortii and PTX detection in scallops, found only PTX-2 in the algae and suggested that oxidation of the 43-methyl group of PTX-2 takes place in the hepatopancreas of the scallop. Studies on the metabolism of PTXs within scallops by Suzuki et al. (1998) indicated that PTX-2 is converted via an oxidation step on the 43-methyl group to PTX-6. This, too, was supported by the fact that PTX-2 was detected in both extracts of D. fortii and in extracts of scallop gut, whereas PTX-6 could only be detected within the gut of the scallop and not in the algal extracts. Another recently identified analogue is pectenotoxin-2 seco acid (PTX-2-SA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2-SA) isolated from greenshell mussels (Daiguji et al., 1998) (Fig. 1). It is characteristic of the PTXs to have a large lactone ring as seen in Fig. 1B, but, unlike these structures, the seco acids have an open chain structure (Fig. 1C). Daiguji et al. (1998) did not find these seco acids
to be cytotoxic towards KB cells at a dose of 1.8 mg/ml, while PTX-2 at a dose of 0.05 mg/ml was cytotoxic. This indicates that the cyclic structure of PTX-2 is required for toxicity. Thus, the conversion of PTX-2 to the seco acid may be protective for detoxification in the shellfish. Suzuki et al. (2001) recently compared the toxin profiles of the seco acids from D. acuta to that of the greenshell and blue mussels isolated from an area when D. acuta was blooming in New Zealand. They found the major PTX toxin in the algae to be PTX-2, whereas both of the shellfish species contained PTX-2-SA and 7-epi-PTX-2-SA (Table 2).
7. Toxicology of the pectenotoxins in mammals The PTXs are hepatotoxic, tumour promoters, and cause apoptosis in rat and salmon hepatocytes (Fladmark et al.,
Table 2 Percentage composition of the different toxins in D. acuta, greenshell mussels, and blue mussels for samples taken in 1998 and in 1996 (taken from Suzuki et al., 2001) Samples (Year of collection) D. acuta (1998) Greenshell mussels (1998) Blue mussels Greenshell mussels (1996) Blue mussels (1996)
Profile (%) PTX-2
PTX-2-SA
7-epi-PTX-2-SA
Other PTXs
92.2 3.1
7.8 86.6
– 10.4
– –
3.8 1.6
88.2 56.6
8.0 41.8
– –
0.9
57.9
41.2
–
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1998). When rat and salmon hepatocytes were dosed with 2 mM PTX-1, apoptotic effects showing extreme nuclear and cellular shrinkage and chromatin hypercondensation were observed within 1 h. The salmon hepatocytes appear more sensitive to the PTXs as demonstrated by the pronounced cell shrinkage that was observed. Cell death was found to be caused by apoptosis rather than by necrosis. This was determined by the observation of a lack of trypan blue uptake and the chromatin condensation typical of apoptosis. In addition, the caspase inhibitor ZVAD-fmk was shown to inhibit the action of PTX-1, which also supports that apoptosis was the cause of cell death. PTX-2 has been found to be potently cytotoxic to the human lung (A-549), colon (HT29), and breast (MCF-7) cancer cell lines, but also cytotoxic to several other cell lines including ovarian, renal, lung, CNS, melanoma, and breast cancer cell lines (Jung et al., 1995). From studies with the DNA cleavage assay and the rat plasma membrane assay, Jung et al. (1995) found that PTX-2 neither exerts its cytotoxic activity by blocking DNA synthesis nor by blocking reduction –oxidation processes within the cell membrane. Pathologically, PTX-1 has been shown to be hepatotoxic causing rapid necrosis of hepatocytes, in particular to periportal regions of hepatic lobules (Terao et al., 1986). Further, PTX was not a causative agent of the diarrhetic symptoms of DSP with dosing levels of 1000, 700, 500, 250, and 150 mg/kg via i.p. (Terao et al., 1986). Mice treated with an intraperitoneal dose of 1000 mg/kg of PTX showed severe congestion of their livers, and the surface of the livers appeared finely granulated within 60 min of the intraperitoneal injection of PTX-1. No pathological changes in the large and small intestines or in any other visceral organs were observable. Multiple large vacuoles appeared around the periportal regions of the lobules. The vacuoles did not stain with neutral red, which indicates changes in neutral lipids, and several of the vacuoles located in the subcapsular region contained erythrocytes. Hyaline droplets were seen in the hepatocytes that developed vacuoles. Electron microscopic observation showed flattening of the microvilli on the hepatocytes in the periportal region and also showed the plasma membrane to be invaginated into its cytoplasm.
These observations in the liver were similar to those observed by Aune (1988) who dosed freshly isolated hepatocytes with various DSPs. More recently, Zhou et al. (1994) used primary cultured liver cells from chick embryos to study the effects of PTX-1 on the cytoskeletons of liver cells with concentrations ranging from 5 ng/ml to 50 mg/ml for 0.5 –24 h. Antitubulin staining revealed that the perinuclear regions had become sparse in microtubules and the remaining perinuclear regions appeared disorganised, while actin microfilaments were also disrupted. Zhou et al. (1994) also found that, for doses up to 5 ng/ml or less, removal of the toxin within 4 h from the culture media revealed that the morphology induced by PTX-1 was reversible. The study by Terao et al. (1986) found that PTX-1 caused damage to the hepatocytes in the periportal area of the lobule. The periportal area approximates to zone 1 of the lobule, which is known to receive blood from the afferent venules and so is rich in oxygen and nutrients (Turton and Hooson, 1998; Timbrell, 1996). Because of this, the hepatocytes in the periportal lobule are more equipped for boxidation of fats and there are also increased levels of glutathione and glutathione reductase in the hepatocytes of this zone. Thus, toxicity seen in this area may indicate that the PTXs are metabolised and removed from the body by a simple conjugation reaction with for example, gluathione or glucuronidation of the hydroxyl groups rather than through a P450 reaction, which is generally located in the zone 3 region and centrilobular damage would be expected if P450 metabolism was the primary metabolic reaction. This finding has been elaborated in a study by Yoon and Kim (1997) where mice were dosed with PTX-2 and a decrease in hepatic microsomal protein content was observed. However, the total P450 content, cytochrome b5, NADPHcytochrome c reductase, and aminopyrine N-demethylase activities were not affected (Yoon and Kim, 1997). Glutathione content was also unaltered, but elevated serum enzyme levels of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase were found (Yoon and Kim, 1997). Changes in these enzymes are known to indicate that hepatocyte damage has occurred (Turton and Hooson, 1998).
Table 3 Summary of toxicity concentrations for various DSP algal toxins in mice Toxin class DSP OA DTX-1 DTX-3 PTX-1 PTX-II PTX-11 PTX1-6 YTX
LD50 toxicity
Reference
Kg Kg Kg Kg Kg Kg Kg
1
100 mg Kg > 1000 mg Kg
1
192 200 166 500 250 260 250
mg mg mg mg mg mg mg
1 1 1 1 1 1
1
i.v. i.v. i.v. i.p. i.p. i.p. i.p.
Tachibana et al., 1981 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Yasumoto et al., 1985 Jung et al., 1995
i.p. p.o
Ogino, 1997
LD99 toxicity 200 mg Kg 160 mg Kg 500 mg Kg
1 1 1
Reference i.p. i.p. i.p.
160 – 170 mg Kg 1 i.p. 100 mg Kg 1 i.p.
Yasumoto and Murata, 1990 Yasumoto and Murata, 1990 Yasumoto and Murata, 1990
Yasumoto and Murata, 1990 Yasumoto and Murata, 1990
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Ishige et al. (1988) conducted an oral toxicity study with PTX-2. At doses ranging from 250 to 2000 mg/kg, PTX-2 caused diarrhetic symptoms accompanied by severe mucosal injuries in the small intestine between 30 and 360 min after dosing by oral injection. Hepatic injuries were also observed as hyaline droplet degeneration in hepatocytes around peripheral zones of the hepatic lobules. Spongiosis lesions of hydropic vacuolar degeneration could be seen in the intermediate zone. Dilatations of the sinusoids and hydropic swelling of the endothelial cells were also observed. The study conducted by Ishige et al. (1988) was one of few oral studies conducted with the PTXs. Levels used in the Ishige et al. (1988) study were very high when compared to what we know of the human situation of shellfish poisoning. This may indicate that humans could be more sensitive to PTX toxins than mice. If this is the case, absorption and metabolism studies must be conducted in the mice to understand the mechanisms of toxicity for the PTXs. Very few studies have been done in mammalian systems that could be directly related to the human situation of shellfish poisoning with regard to the metabolism of the PTXs. With reference to Table 3, it can be seen that, in mice, the LD50 toxicity of PTX-1 and PTX-2 has been determined to be 250 mg/kg, ip, for PTX-1 and 260 mg/kg, ip, for PTX-2. These figures are higher than the LD50 value for DTX-3 (500 mg/kg) but lower toxicity than that seen for DTX-1 (166 mg/kg) or OA at 200 mg/kg. The highest toxicity for DSP toxins is seen with the YTXs with an i.p. LD50 of 100 mg/kg. By the oral route, yessotoxin was not found to be toxic at 1000 mg/kg. It has been found that the oral toxicity of OA is 25– 50 times lower than that seen from i.p. dosing (Aune et al., 1998). A review article by Yasumoto and Murata (1990) stated that the LD99 in mice (intraperitoneal) for PTX-1 –6 ranged from 106 to 770 mg/kg. This study did not define which PTX isomer caused the greatest toxicity, and did not provide detail of any of the pathologies observed. LD50 values are useful in providing information for regulatory and safety assessments and indicating the potency of the toxin but provide no information on the mechanisms of toxicity and other pathological processes that would aid in the development of antidotes or treatments for the toxins.
8. Pectenotoxins and public health All the in vivo studies with the PTXs discussed above have used very high dosing levels compared with the doses known to cause illness in the human situation. PTXs have been associated with severe diarrhetic illness resulting in hospitalisation. One such incident occurred during December of 1997 when over 100 people living in the Eastern states of Australia were poisoned following consumption of pipis harvested off the New South Wales (NSW) coastline. This particular incident included 56 cases of hospitalisation in NSW alone. Symptoms included nausea, vomiting, and
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diarrhoea. The shellfish responsible for the poisoning were found to contain PTX-2-SA and low levels of PTX-2 (Eaglesham et al., 2000). Another poisoning incident involving the consumption of pipis (Donax delatoides) occurred on North Stradbroke Island (QLD, Australia) in March 2000 where an elderly woman became seriously ill after eating pipis from one of the local beaches. To investigate this incident, fresh pipis were collected from a pristine beach on the island with no sewage outlets. High levels of PTX-2-SA were found in the shellfish that caused the illness (Eaglesham et al., 2000). Pipis can metabolise PTX-2 to PTX-2-SA which explains the different toxin profiles seen between the shellfish and the phytoplankton at the time of the Stradbroke poisoning. Thus, it seems possible that these seco acids are responsible for the diarrhetic symptoms seen or that once ingested, they may convert back to PTX-2 prior to absorption in to the body perhaps in the strongly acid or alkaline environment of the digestive tract. Alternatively, the toxin may be absorbed as the seco acid and metabolised in the liver. No toxicity data has been published on the PTX-2 seco acid isomers to date but toxicopathologies are currently under investigation (Burgess et al., 2001). By analysis of the pipis from the poisoning incident, the amount of toxin consumed by the patient was estimated. It was found that the pipis contained approximately 300 mg/kg of pipis. The average meal consists of approximately 0.5 kg of pipis. Therefore, the person consumed approximately 150 mg of PTX-2-SA during the meal. For an average 70-kg person, this equates to a dose of 2 mg PTX-2-SA/kg body weight leading to illness in humans. This figure is considerably lower than the doses used in the toxicity studies in mice described in this review. The amount of DTX-1 sufficient to induce gastroenteritis in a human is 32 mg when eaten (Yasumoto et al., 1985). This also, is a very low figure compared to the doses used in the toxicology studies for DTXs described in this article. In light of this information, the question should be posed as to why the oral toxicity studies are conducted with such high doses in mice? In addition, the question is raised ‘‘are humans more sensitive to DSP toxins than mice?’’ The symptoms experienced at lower doses may relate to differences in metabolism for the DSPs or other physiological processes such as absorption mechanisms and simply to differences between intestinal surface area to body weight ratios. It is fundamental to our understanding of the process of poisoning to gather information on the mechanisms of toxicity of these toxins. It is not possible to conduct an accurate health-risk assessment without such information.
9. Conclusions The PTXs are known to cause severe acute illness in humans. Research has shown that these toxins are potently cytotoxic, tumour promoters, and cause necrosis to hepato-
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cytes. Nothing is known of the chronic toxicology of PTXs or the potential implications to public health in the long term. This information is needed to perform health-risk assessments for consumption of contaminated shellfish, and for guidance in the regulation of permitted toxin levels in shellfish that are sold for human consumption. It is evident, for an appropriate health-risk assessment to be performed, more toxicology and metabolism work is required on the PTXs, especially where the oral toxicity is concerned. Research must be conducted with relevance to the human situation of shellfish poisoning. It is also important that research is undertaken to assess the effects of consuming several of the DSP toxins in one meal, as often occurs in the real-life situation where shellfish can be contaminated with a combination of DSP toxins.
Acknowledgments NRCET is funded by the National Health and Medical Research Council, Queensland Health, Griffith University, Queensland University of Technology and the University of Queensland.
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