- Email: [email protected]
Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneus
Toxicon 40 (2002) 1463–1469 www.elsevier.com/locate/toxicon
Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneusq L.E. Llewellyna,*, M.J. Doddb, A. Robertsona, G. Ericsona, C. de Koningb, A.P. Negria b
a Australian Institute of Marine Science, PMB 3, Townsville MC, Qld. 4810, Australia Victorian Institute of Forensic Medicine, Monash University, 57-83 Kavanagh Street, Southbank, Vic. 3006, Australia
Received 8 March 2002; accepted 29 May 2002
Abstract After ingestion of a specimen of the crab Zosimus aeneus (Xanthidae), an East Timorese adult male died within several hours. Xanthid crabs are known to harbour paralytic shellfish toxins (PSTs), tetrodotoxin and palytoxin. A post-mortem examination did not find any obvious pathological abnormalities. This absence of pathologies is more often associated with PSTs and tetrodotoxin intoxication. A second, yet uneaten specimen of Z. aeneus from the same meal, contained a significant amount of PSTs and these same toxins were identified in the gut contents, blood, liver and urine of the victim. Metabolism of the PSTs occurred with the ingested crab harbouring gonyautoxin 2, gonyautoxin 3 and saxitoxin (STX) whereas neoSTX, decarbamoylSTX and STX dominated the PSTs in the victim’s urine. The PST composition in the gut contents, in both their identity and proportion, was intermediate between the eaten crab and the urine suggesting that toxin conversion commenced in the victim’s gut. The dose consumed by the victim was calculated to be between 1 and 2 mg STX equivalents/kg based upon the concentration in the remains of the cooked crab. The victim’s meal did not consist solely of the toxic crab eaten and the possibility of other food items acting in a synergistic manner with the consumed PSTs cannot be discounted. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Zosimus aeneus; Saxitoxin; Sodium channel; Saxiphilin; Receptor bioassay; Crab poisoning; Fatality; East Timor
1. Introduction Human fatalities from crab ingestion, usually of the family Xanthidae, have been documented from Japan (Hashimoto et al., 1967), Philippines (Alcala et al., 1988; Alcala and Halstead, 1970; Gonzales and Alcala, 1977), Fiji (Raj et al., 1983), Palau Islands (Mote et al., 1978), Mauritius (Halstead and Cox, 1973) and Vanuatu (Llewellyn, 2001). Xanthid crabs can harbour paralytic shellfish toxins (PSTs) (Anderson, 1994), tetrodotoxin (Tsai et al., 1995) and a palytoxin-like substance (Alcala q This is publication number 1096 of the Australian Institute of Marine Science. * Corresponding author. Tel.: þ61-7-4753-4449; fax: þ 61-74772-5852. E-mail address: [email protected] (L.E. Llewellyn).
et al., 1988), with PSTs being by far the most common of these three toxin groups found in xanthid crabs. We report here a human fatality which occurred after eating a specimen of the xanthid crab Zosimus aeneus previously reported to harbour PSTs (Arakawa et al., 1994; Llewellyn and Endean, 1989; Raj et al., 1983) and tetrodotoxin (Hwang et al., 1996). Another species of xanthid crabs, namely Lophozozymus pictor, has been found to occasionally have palytoxin in its flesh (Alcala et al., 1988; Lau et al., 1995). PST-like bioactivity was detected using the sodium channel and saxiphilin radioreceptor assays (Doucette et al., 1997; Llewellyn et al., 2001a,b) and confirmation of assay results was achieved by HPLC analysis. This allowed us to identify PSTs in the victim’s gut contents and throughout various body fluids and tissues obtained post-mortem from the victim.
0041-0101/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 1 6 4 - 2
1464
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
2. Materials and methods 2.1. Materials All buffers and general chemicals were from Sigma (St Louis, MO, USA) and water was deionised (<18 MV) with a Barnstead system (Barnstead, IA, USA). Saxiphilin from the centipede Ethmostigmus rubripes and rat brain synaptosomes were prepared and stored as reported previously (Llewellyn, 1997; Llewellyn et al., 1998). Tritiated saxitoxin ([3H]STX) was purchased from Amersham Pharmacia Biotech (UK). Saxitoxin dihydrochloride (STX) (Institute for Marine Biosciences, National Research Council, Canada) was diluted in 1 mM citrate buffer (pH 5.0). Toxin standards for HPLC analysis of PSTs were kindly donated by Prof. Y. Oshima and Dr H. Onodera of Tohoku University, Japan. 2.2. Post-mortem samples and their extraction Liver, blood, bile, gut contents and urine were removed post-mortem and along with the cooked uneaten remains of crabs were stored frozen until extracted for toxin analysis. Blood was separated into plasma and cells after the addition of heparin (1:5, v/v 100 U/ml heparin in phosphate buffered saline composing 136 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and centrifuging at 4300g for 15 min. Gut contents were also separated into soluble and insoluble fractions by centrifuging at 20,000g for 20 min. Fluid samples (1 ml) (i.e. bile, urine, plasma, gut contents supernatant) were extracted by dilution (1:1, v/v) with 0.1 M acetic acid (final [acetic acid] ¼ 0.05 M). This mixture was then applied to a 500 mg Extract-Clean C18 sep-pak (Alltech Associates, NSW, Australia) preequilibrated with 10 ml methanol, followed by 20 ml of 0.05 M acetic acid. The sample was applied and the eluate collected. The sep-pak was then washed with 5 ml 0.05 M acetic acid that was also collected. Treatment of human forensic samples for PST analysis with reverse phase seppak treatment has been shown previously not to cause any loss of toxin from such samples (Gessner et al., 1997; Powell and Doucette, 1999). Attached to the end of the seppak was 0.2 mm filter (Minisart, Sartorius, Gottingen, Germany). This eluate was then dried in vacuo and the sample redissolved into 1 ml 0.05 M acetic acid, this being the same as the original starting volume. This sample was then passed through a 10,000 MWCO centrifugal ultrafilter (Ultrafree-MC, Millipore, Sydney, Australia) to yield the sample used for bioassay analysis (cold extract). An aliquot of this ultrafiltrate was then dried in vacuo and redissolved in an equal volume of 0.1N HCl and subjected to 90 8C for 5 min (AOAC extract). Solid samples (crabs, blood cells, liver and gut contents pellet) were extracted with a slightly modified protocol to that recommended by the AOAC for PST extraction and
testing. Instead of adding 1 ml of extraction solvent to each gram of tissue, 5 ml of 0.1N HCl was added prior to homogenisation using a Heidolph tissue disruptor and heating the mixture at 90 8C for 5 min. The extract was clarified by centrifugation at 20,000g for 20 min (4 8C). Supernatant was decanted and passed through a 10,000 MWCO centrifugal ultrafilter to give the sample used for analysis (AOAC extract). Unheated extracts of these samples were also performed following the above procedure except that the solvent was 80% ethanol acidified to pH 2.0 with HCl, with the samples kept on ice or at 4 8C at all times (cold extract). 2.3. Radioreceptor assays for paralytic shellfish toxins The saxiphilin [3H]STX binding and voltage-gated sodium channel (Na channel) binding assays were conducted as previously described (Llewellyn et al., 2001a,b) with the exception of using 5 nM [3H]STX. Saxitoxin concentration equivalents (STXeq) was determined by back-calculation from calibration curves using standard STX after measuring [3H]STX inhibition by extracts at a series of dilutions as explained in detail elsewhere (Llewellyn et al., 2001a,b). Calibration curves generated with standard STX were ((100 2 F )/F )0.9 £ 4.4 nM for Na channel and ((100 2 F )/F )1.2 £ 6.3 nM for saxiphilin, where F ¼ %[3H]STX bound in the presence of extracts relative to controls, n and IC50 were the Hill slope and concentration of STX which causes 50% inhibition in the calibration curves, respectively. 2.4. Chromatographic analysis for paralytic shellfish toxins Selected samples were lyophilised and redissolved into 1 ml Milli-Q purified water to be then fractionated using a Sephadex G-15 column (1.5 £ 20 cm2, Amersham Pharmacia Biotech, Sydney, Australia) equilibrated in Milli-Q purified water. The column was eluted with 30 ml water, followed successively by 30 ml 0.005 M acetic acid, 30 ml 0.01 M acetic acid, 30 ml 0.025 M acetic acid, 30 ml 0.05 M acetic acid, and finally by 30 ml of 0.1 M acetic acid. Fractions (5– 10 ml) were collected, lyophilised and the residue resuspended into 0.05 M acetic acid whereupon they were assayed in the Na channel assay as described earlier for the detection of [3H]STX displacing activity. Active fractions were then subjected to standard HPLC analysis and compared to toxin standards (Llewellyn et al., 2001a,b; Oshima, 1995). HPLC toxin profiles were converted to absolute toxin values by multiplying the concentration of each toxin in an extract by its toxicity relative to STX (Oshima, 1995) and summing to obtain the total extract toxicity in STXeq. Toxins were identified by comparison of retention times and fluorescence emission maxima with standards, the disappearance of peaks by eliminating postcolumn oxidation and spiking experiments.
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
1465
Table 1 Quantitation of total PST load of extracts of crabs and human post-mortem samples using Na channel and saxiphilin radioreceptor assays Sample
Extract type
Na channel (mg STXeq/100 ml or 100 g tissue)
Saxiphilin (mg STXeq/100 ml or 100 g tissue)
Z. aeneus individual 1
Colda AOACb Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC
76.7 ^ 14.2 461.5 ^ 92.7 24.2 ^ 1.0 289.6 ^ 12.9 12.1 ^ 2.2 15.8 ^ 2.6 0.9 ^ 0.4 0.9 ^ 0.3 Not detected Not detected 4.4 ^ 1.3 4.2 ^ 1.4 Not detected 10.3 ^ 1.0 423.4 ^ 119.7 577.1 ^ 58.9 448.4 ^ 83.1 385.4 ^ 92.9
331.2 ^ 52.8 291.6 ^ 6.2 111.8 ^ 10.9 346.7 ^ 83.8 10.0 ^ 2.7 10.1 ^ 3.4 4.9 ^ 1.8 4.0 ^ 0.1 Not detected Not detected 4.1 ^ 1.8 8.9 ^ 3.2 Not detected Not detected 260.8 ^ 48.6 257.5 ^ 40.2 224.1 ^ 14.8 376.2 ^ 37.8
Z. aeneus individual 2 Urine Liver Bile Blood plasma Blood cells Gut contents pellet Gut contents supernatant a b
Cold extraction method described in Section 2.2. Extracted by modified AOAC extraction method, which uses heat and acid.
3. Results 3.1. Preamble, case report and post-mortem In 1999, United Nations personnel were located in East Timor during a time of political instability and as part of their duties, they undertook investigations of suspected criminal cases. One of the authors (MJD) was consultant forensic pathologist and was asked to examine the body of a slightly built but well nourished 32 year old East Timorese male found dead on Behau Beach on 1 October 2000, after participating in a party. An initial suspicion that the deceased was murdered precipitated a full forensic examination. Police investigation revealed the victim had collected two crabs and another marine creature tentatively identified as being a member of the Class Holothuroidea (Phylum Echinodermata) at about 9.30 a.m. After cooking the animals on an open fire, part of one of the crabs and the holothurian were eaten. The victim rejoined the party, reportedly ‘ran around’ and drank two cans of alcoholic beer. Soon after, the victim vomited and lay down to rest at 11.00 a.m., stating he was ‘exhausted’. An hour later, it was discovered that he was dead. Nothing remarkable was observed upon external examination of the victim. Internal examination did not reveal any obvious disease. The victim had moderate pulmonary congestion and oedema and marked congestion of the meningeal vessels but no other abnormalities were observed, especially of the heart, coronary arteries and brain. No illicit drugs or medications were detected by routine toxicological analysis. Histological samples of all tissues were prepared and again revealed no
significant abnormalities apart from confirming non-specific congestion and oedema. The uneaten crab and the remains of the crab that was partly consumed were both identified by Dr Gary Poore, Curator of Crustacea, Department of Zoology, University of Melbourne as Z. aeneus, a crab species from the family Xanthidae previously implicated in fatal human intoxications. The absence of obvious pathological and histological abnormalities indicated that palytoxin did not contribute to the victim’s death. This toxin is hemorrhagic to mammals and mediates toxicity through significant effects on cardiovascular, kidney, gastrointestinal and respiratory systems (Wiles et al., 1974). This can result in congestion of organs and histological abnormalities are apparent in liver, kidney, heart, pancreatic and intestinal tissues (Terao et al., 1992). All remaining post-mortem samples were therefore analysed for toxins with the background knowledge that primarily PSTs, but on occasion, tetrodotoxin have been detected from this species of crab (Arakawa et al., 1994; Hwang et al., 1996; Llewellyn and Endean, 1989; Raj et al., 1983). 3.2. Screening and quantitation with radioreceptor assays Table 1 lists the PST concentrations (STXeq) calculated from Na channel and saxiphilin inhibition results from the various post-mortem extracts. STXeq values were calculated from the nanomolar values derived from calibration curves using the molecular weight of STX2þ of 301 and the sample dilution factors. Statistical comparison of the two datasets was undertaken using the Student’s t-test and by calculating the Spearman’s correlation coefficient, treating
1466
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
Table 2 Toxin profiles of Z. aeneus individual 2 and the victim’s urine and gut content supernatant samples calculated from HPLC chromatograms. ND signifies that the peak could not be positively identified as dcSTX Toxin profiles (mol%)
Z. aeneus individual 2 Gut content supernatant Urine
Total toxicity (mg STXeq/100 ml or 100 g tissue)
GTX2
GTX3
NEO
dcSTX
STX
45.5 5.5 0.1
17.3 10.2 0.3
0 2.6 9.6
0 ND 41.3
37.2 81.8 48.7
those samples in which toxin was not detected as zero. A two-tailed paired t-test generated a t-statistic of 0.92, which corresponds to a probability value of 0.37 (df ¼ 17). The Spearman’s correlation coefficient was 0.83 ( p , 0.0005). The values obtained by these two assay methods are therefore not statistically different and closely correlated. 3.3. Chromatography and HPLC confirmation of PSTs from samples HPLC analysis was performed on the cold acetic acid extracts that contained enough toxin for confident analysis (i.e. uneaten crab, stomach content and urine samples), where the original toxin profiles would be maintained (Fig. 1). To remove interfering fluorescent compounds, it was necessary to fractionate the samples using Na channel guided fractionation on a Sephadex G-15 column. The uneaten crab contained STX (37%) and the singly sulphated toxin epimers GTX2 (46%) and GTX3 (17%) (Table 2). No C-class toxins (i.e. doubly sulphated PSTs) were detected in any samples. The gut content supernatant was dominated by STX (82%), with lesser amounts of neoSTX (almost 3%) and GTX2 (6%) and GTX3 (10%). dcSTX could not be positively confirmed in this sample due to the presence of interfering fluorescent peaks. The toxin profile of the victim’s urine sample, however, comprised only 50% STX and much lower levels of GTX2 (0.1%) and GTX3 (0.3%). Compared to the gut contents, there was an increased proportion of the neoSTX (10%) and dcSTX was clearly resolved and prominent comprising 41% of the urine toxin profile. The total toxin concentration of the uneaten crab was 163 mg STXeq/100 g (Table 2), higher than the toxicity detected in each assay of the cold acetic acid extracts. The toxicity detected in the urine sample (15 mg STXeq/100 ml) agreed with both of the assays whereas HPLC detected a lower level of toxicity in the gut (30 mg STXeq/100 ml) compared with the assay techniques.
4. Discussion It is not surprising the victim perished after eating a Z. aeneus crab. Between 65 and 100% of Z. aeneus crabs
162.8 29.9 15.3
collected from Japan, Philippines, Australia and Fiji were toxic as measured by mouse lethality bioassay (Koyama et al., 1983; Llewellyn and Endean, 1989; Negri and Llewellyn, 1998; Raj et al., 1983; Yasumura et al., 1986). Further still, many of the reported toxicity values for these crabs exceeded the 80 mg STXeq/100 g tissue limit widely used by regulators to prohibit harvesting of molluscan shellfish for human consumption (Van Egmond et al., 1992). For example, by using the conversion factor of 1 mouse unit equalling 0.18 mg STX (Johnson and Mulberry, 1966), the proportion of crabs that contained more than 80 mg STXeq/100 g tissue ranged from 44% from Fijian specimens (Raj et al., 1983) to 100% in the Philippines and Japan (Koyama et al., 1983). The Na channel and saxiphilin receptor assays have been shown to closely correlate to mouse bioassay quantifications (Doucette et al., 1997; Llewellyn et al., 2001a,b). The fact that saxiphilin is insensitive to TTX whereas the Na channel is sensitive to both PSTs and TTX has been used to differentiate extracts containing significant amounts of TTX (Negri and Llewellyn, 1998). The close and statistically significant agreement between the two radioreceptor assays in Table 1 indicate that TTX is an insignificant contributor to the crab’s toxicity. Toxicity calculated from summing individual components from HPLC traces agreed well with the receptor assays. In the gut supernatant, however, a lower toxicity was reported by HPLC. Toxin peaks may have been masked by unremoved interfering compounds. It is also possible that some of the fluorescent peaks detected may have been undescribed PST analogues that contributed to the toxicity measured by bioassay. Small amounts of toxicity were detected in blood, liver and urine. This agrees with previous results where PST elimination in molluscan intoxicated humans was primarily via urination (Gessner et al., 1997; Montebruno, 1993). The presence in blood confirms the pathway by which the toxin is distributed about the body with some toxin being resident in the liver as has been observed before with Chilean molluscan PSP victims (Montebruno, 1993). The PST profile in the gut supernatant was dominated by STX whereas the urine contained only 50% STX. The increased proportion of neoSTX in the victim’s urine indicates that oxidation of the N1 group may be an important mechanism of elimination (Eq. (1)). The urine sample also
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
1467
Fig. 1. Several examples of HPLC analysis of PSTs from samples analysed in this study. Trace A, C and E are using conditions targeting gonyautoxins 1–5 with A being toxin standards, C being of the toxic fractions from crab #2 and E is of a toxic urine fraction. Traces B, D and F are of analyses targeting STX, dcSTX and neoSTX with B being toxin standards, trace D being of the toxic fractions from crab #2 and trace F is of the same toxic urine fraction as in E. Peaks labelled non-PSTs elute at similar retention times as standards but were shown not to be PSTs by their disappearance after eliminating post-column oxidation and by spiking experiments.
contained high proportions of dcSTX, indicating hydrolysis of the carbamoyl group of STX to form its decarbamoyl analogue (Eq. (2)). 1. Oxidation of N1 group to form the hydroxy analogue STX ! neoSTX
ð1Þ
2. Hydrolysis of carbamoyl group to form decarbamoyl analogue (Cembella et al., 1993; Oshima, 1996;
Sullivan et al., 1983) STX ! dcSTX
ð2Þ
The uneaten remains of the consumed crab weighed only 10 g. Less than half of the crab was eaten so no more than 10 g of tissue was ingested. Using the maximum toxicity determination, that for the gut content pellet that had undergone AOAC extract preparation, the victim could not have eaten more than 60 mg STXeq. Taking into
1468
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
account the victim’s weight of approximately 50 kg, this gives a lethal oral dose of 1–2 mg STXeq/kg. This is less than lethal doses previously reported for PSP which have ranged from as low as 6 mg STXeq/kg (Shumway, 1995) to 4375 mg STXeq/ kg (Rodrigue et al., 1990). It is unlikely that significant levels of TTX were present in the samples, based upon the correlation between the PST specific saxiphilin assay and the PST and TTX sensitive Na channel assays, the amount of TTX present would add little to overall toxin burden consumed by the victim. It is worth noting that the crab flesh eaten in this case may have contained more toxicity than the value reported here for the whole crabs. For example the victim’s gut content had a higher relative toxicity than the remains of the meal itself. It has previously been reported that toxicity levels can vary greatly between different tissues of Z. aeneus. For example, again using the conversion factor of 1 mouse unit equalling 0.18 mg STX (Johnson and Mulberry, 1966), the muscle of the cephalothorax had 720 mg STXeq/100 g tissue while the muscle from the chelae had 108,000 mg STXeq/100 g tissue (Konosu et al., 1969). The range of countries where crab ingestion has killed people now includes East Timor. It is quite likely that crab poisonings have occurred previously in East Timor as anecdotal reports to United Nations field medical staff revealed that suicides were occasionally achieved by the intentional consumption of known toxic crabs, indicating a local knowledge of the potential toxicity of these crabs. The likelihood of victims receiving medical aid is low considering that the time until death after ingestion can be extraordinarily short. In this case, the victim was dead within several hours of eating the crabs, an interval between ingestion and death reported previously for victims of crab (Hashimoto et al., 1967; Mote et al., 1978) and molluscan induced PSP (Rodrigue et al., 1990). Bearing in mind the complexity of a meal and the variety of means by which they can be prepared, one cannot discount the possibility that other compounds within the victim’s meal may have promoted toxin uptake, a worrisome consideration for those with a public health interest in these naturally occurring toxins. The detection of PSTs associated with commercial seafood in temperate waters such as mussels and clams is routine; however, the recreational fishing of crabs in the tropics remains largely unregulated. This type of activity, coupled with a lack of scientific infrastructure in the tropics, means that the number of PST victims from crab poisoning is likely to be underestimated. Only greater public and practitioner awareness of potentially toxic species and symptoms and the development of rapid, cost effective testing kits will result in a decrease in PST related fatalities in this region.
References Alcala, A., Halstead, B., 1970. Human fatality due to ingestion of the crab Demania sp. in the Philippines. Clin. Toxicol. 3, 609–611.
Alcala, A., Alcala, L., Garth, J., Yasumura, D., Yasumoto, T., 1988. Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon 26, 105–107. Anderson, D.M., 1994. Red tides. Sci. Am. 271, 62 –68. Arakawa, O., Noguchi, T., Shida, Y., Onoue, Y., 1994. Occurrence of carbamoyl-N-hydroxy derivatives of saxitoxin and neosaxitoxin in a xanthid crab Zosimus aeneus. Toxicon 32, 175 –183. Cembella, A., Shumway, S., Lewis, N., 1993. Anatomical distribution and spatio-temporal variation in paralytic shellfish toxin composition in two bivalve species from the Gulf of Maine. J. Shellfish Res. 12, 389–403. Doucette, G., Logan, M., Ramsdell, J., Van Dolah, F., 1997. Development and preliminary validation of a microtiter platebased receptor binding assay for paralytic shellfish poisoning toxins. Toxicon 35, 625– 636. Gessner, B.D., Bell, P., Doucette, G.J., Moczydlowski, E., Poli, M.A., Van Dolah, F., Hall, S., 1997. Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks. Toxicon 35, 711 –722. Gonzales, R.B., Alcala, A.C., 1977. Fatalities from crab poisoning on Negros Island, Phillippines. Toxicon 15, 169–170. Halstead, B.W., Cox, K.W., 1973. A fatal case of poisoning by the red-spotted crab Carpilius maculatus (Linnaeus) in Mauritius. Proc. R. Soc. Arts Sci. Mauritius 4, 27 –30. Hashimoto, Y., Konosu, S., Yasumoto, T., Inoue, A., Noguchi, T., 1967. Occurrence of toxic crabs in Ryukyu and Amami Islands. Toxicon 5, 85–90. Hwang, D.F., Tsai, Y.H., Chai, T.J., Jeng, S.S., 1996. Occurrence of tetrodotoxin and paralytic shellfish poison in Taiwan crab Zosimus aeneus. Fish. Sci. 62, 500–501. Johnson, H., Mulberry, G., 1966. Paralytic shellfish poison: serological assay by passive haemagglutination and bentonite flocculations. Nature 211, 747 –748. Konosu, S., Inoue, A., Noguchi, T., Hashimoto, Y., 1969. A further examination on the toxicity of three species of xanthid crabs. Nippon Suisan Gakkaishi 35, 88– 92. Koyama, K., Noguchi, T., Uzu, A., Hashimoto, K., 1983. Individual, local, and size-dependent variations in toxicity of the xanthid crab Zosimus aeneus. Nippon Suisan Gakkaishi 49, 1273–1279. Lau, C., Tan, C., Li, Q., Ng, F., Yuen, R., Khoo, H., 1995. Bioactivity and mechanism of action of Lophozozymus pictor toxin. Toxicon 33, 901– 908. Llewellyn, L.E., 1997. Haemolymph protein in xanthid crabs: its selective binding of saxitoxin and possible role in toxin bioaccumulation. Mar. Biol. 128, 599 –606. Llewellyn, L.E., 2001. Human fatalities in Vanuatu after ingestion of a crab, Daira perlata. Med. J. Aust. 175, 343– 344. Llewellyn, L., Endean, R., 1989. Toxicity and paralytic shellfish toxin profiles of the xanthid crabs, Lophozozymus pictor and Zosimus aeneus, collected from some Australian coral reefs. Toxicon 27, 596–600. Llewellyn, L.E., Doyle, J., Negri, A., 1998. A high throughput, microtitre plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin. Anal. Biochem. 261, 51 –56. Llewellyn, L.E., Doyle, J., Jellett, J., Barrett, R., Alison, C., Bentz, C., Quilliam, M., 2001a. Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469 saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay. Food Add. Contam. 18, 970 –980. Llewellyn, L.E., Negri, A.P., Doyle, J., Baker, P.D., Beltran, E.C., Neilan, B.A., 2001b. Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis. Env. Sci. Tech. 35, 1445–1451. Montebruno, D., 1993. Paralytic shellfish poisoning in Chile. Med. Sci. Law 33, 243–246. Mote, G.E., Halstead, B.W., Hashimoto, Y., 1978. Occurrence of toxic crabs in the Palau Islands. Clin. Toxicol. 3, 579–607. Negri, A., Llewellyn, L., 1998. Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia. Toxicon 36, 283 –298. Oshima, Y., 1995. Post-column derivatization HPLC methods for paralytic shellfish poisons. In: Hallegraeff, G., Anderson, D., Cembella, A. (Eds.), Manual on Harmful Marine Microalgae, Intergovernmental Oceanographic Commission Manuals and Guides, 33. UNESCO, Paris, pp. 81– 94. Oshima, Y., 1996. Chemical and enzymatic transformation of paralytic shellfish toxins in marine organisms. In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (Eds.), Harmuful and Toxic Algal Blooms, Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 475 –480. Powell, C.L., Doucette, G.J., 1999. A receptor binding assay for
1469
paralytic shellfish poisoning toxins: recent advances and applications. Nat. Toxins 7, 393–400. Raj, U., Haq, H., Oshima, Y., Yasumoto, T., 1983. The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands. Toxicon 21, 547– 551. Rodrigue, D., Etzel, R., Hall, S., de Porras, E., Velasquez, O., Tauxe, R., Kilbourne, E., Blake, P., 1990. Lethal paralytic shellfish poisoning in Guatemala. Am. J. Trop. Med. Hyg. 42, 267 –271. Shumway, S., 1995. Phycotoxin-related shellfish poisoning: bivalve molluscs are not the only vectors. Rev. Fish. Sci. 3, 1–31. Sullivan, J., Iwaoka, W., Liston, J., 1983. Enzymatic transformation of PSP toxins in the Littleneck Clam (Protothaca staminea ). Biochem. Biophys. Res. Commun. 114, 465 –472. Terao, K., Ito, E., Yasumoto, T., 1992. Light and electron microscopic observation of experimental palytoxin poisoning in mice. Bull. Soc. Pathol. Exot. 85, 494–496. Tsai, Y., Hwang, D., Chai, T., Jeng, S., 1995. Occurrence of tetrodotoxin and paralytic shellfish poison in the Taiwanese crab Lophozozymus pictor. Toxicon 33, 1669–1673. Van Egmond, H., Speijers, G., Van den Top, H., 1992. Current situation on worldwide regulations for marine phycotoxins. J. Nat. Toxins 1, 67– 85. Wiles, J., Vick, J., Christensen, M., 1974. Toxicological evaluation of palytoxin in several animal species. Toxicon 12, 427 –433. Yasumura, D., Oshima, Y., Yasumoto, T., Alcala, A., Alcala, L., 1986. Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric. Biol. Chem. 50, 593– 598.
Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneusq L.E. Llewellyna,*, M.J. Doddb, A. Robertsona, G. Ericsona, C. de Koningb, A.P. Negria b
a Australian Institute of Marine Science, PMB 3, Townsville MC, Qld. 4810, Australia Victorian Institute of Forensic Medicine, Monash University, 57-83 Kavanagh Street, Southbank, Vic. 3006, Australia
Received 8 March 2002; accepted 29 May 2002
Abstract After ingestion of a specimen of the crab Zosimus aeneus (Xanthidae), an East Timorese adult male died within several hours. Xanthid crabs are known to harbour paralytic shellfish toxins (PSTs), tetrodotoxin and palytoxin. A post-mortem examination did not find any obvious pathological abnormalities. This absence of pathologies is more often associated with PSTs and tetrodotoxin intoxication. A second, yet uneaten specimen of Z. aeneus from the same meal, contained a significant amount of PSTs and these same toxins were identified in the gut contents, blood, liver and urine of the victim. Metabolism of the PSTs occurred with the ingested crab harbouring gonyautoxin 2, gonyautoxin 3 and saxitoxin (STX) whereas neoSTX, decarbamoylSTX and STX dominated the PSTs in the victim’s urine. The PST composition in the gut contents, in both their identity and proportion, was intermediate between the eaten crab and the urine suggesting that toxin conversion commenced in the victim’s gut. The dose consumed by the victim was calculated to be between 1 and 2 mg STX equivalents/kg based upon the concentration in the remains of the cooked crab. The victim’s meal did not consist solely of the toxic crab eaten and the possibility of other food items acting in a synergistic manner with the consumed PSTs cannot be discounted. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Zosimus aeneus; Saxitoxin; Sodium channel; Saxiphilin; Receptor bioassay; Crab poisoning; Fatality; East Timor
1. Introduction Human fatalities from crab ingestion, usually of the family Xanthidae, have been documented from Japan (Hashimoto et al., 1967), Philippines (Alcala et al., 1988; Alcala and Halstead, 1970; Gonzales and Alcala, 1977), Fiji (Raj et al., 1983), Palau Islands (Mote et al., 1978), Mauritius (Halstead and Cox, 1973) and Vanuatu (Llewellyn, 2001). Xanthid crabs can harbour paralytic shellfish toxins (PSTs) (Anderson, 1994), tetrodotoxin (Tsai et al., 1995) and a palytoxin-like substance (Alcala q This is publication number 1096 of the Australian Institute of Marine Science. * Corresponding author. Tel.: þ61-7-4753-4449; fax: þ 61-74772-5852. E-mail address: [email protected] (L.E. Llewellyn).
et al., 1988), with PSTs being by far the most common of these three toxin groups found in xanthid crabs. We report here a human fatality which occurred after eating a specimen of the xanthid crab Zosimus aeneus previously reported to harbour PSTs (Arakawa et al., 1994; Llewellyn and Endean, 1989; Raj et al., 1983) and tetrodotoxin (Hwang et al., 1996). Another species of xanthid crabs, namely Lophozozymus pictor, has been found to occasionally have palytoxin in its flesh (Alcala et al., 1988; Lau et al., 1995). PST-like bioactivity was detected using the sodium channel and saxiphilin radioreceptor assays (Doucette et al., 1997; Llewellyn et al., 2001a,b) and confirmation of assay results was achieved by HPLC analysis. This allowed us to identify PSTs in the victim’s gut contents and throughout various body fluids and tissues obtained post-mortem from the victim.
0041-0101/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 1 6 4 - 2
1464
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
2. Materials and methods 2.1. Materials All buffers and general chemicals were from Sigma (St Louis, MO, USA) and water was deionised (<18 MV) with a Barnstead system (Barnstead, IA, USA). Saxiphilin from the centipede Ethmostigmus rubripes and rat brain synaptosomes were prepared and stored as reported previously (Llewellyn, 1997; Llewellyn et al., 1998). Tritiated saxitoxin ([3H]STX) was purchased from Amersham Pharmacia Biotech (UK). Saxitoxin dihydrochloride (STX) (Institute for Marine Biosciences, National Research Council, Canada) was diluted in 1 mM citrate buffer (pH 5.0). Toxin standards for HPLC analysis of PSTs were kindly donated by Prof. Y. Oshima and Dr H. Onodera of Tohoku University, Japan. 2.2. Post-mortem samples and their extraction Liver, blood, bile, gut contents and urine were removed post-mortem and along with the cooked uneaten remains of crabs were stored frozen until extracted for toxin analysis. Blood was separated into plasma and cells after the addition of heparin (1:5, v/v 100 U/ml heparin in phosphate buffered saline composing 136 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and centrifuging at 4300g for 15 min. Gut contents were also separated into soluble and insoluble fractions by centrifuging at 20,000g for 20 min. Fluid samples (1 ml) (i.e. bile, urine, plasma, gut contents supernatant) were extracted by dilution (1:1, v/v) with 0.1 M acetic acid (final [acetic acid] ¼ 0.05 M). This mixture was then applied to a 500 mg Extract-Clean C18 sep-pak (Alltech Associates, NSW, Australia) preequilibrated with 10 ml methanol, followed by 20 ml of 0.05 M acetic acid. The sample was applied and the eluate collected. The sep-pak was then washed with 5 ml 0.05 M acetic acid that was also collected. Treatment of human forensic samples for PST analysis with reverse phase seppak treatment has been shown previously not to cause any loss of toxin from such samples (Gessner et al., 1997; Powell and Doucette, 1999). Attached to the end of the seppak was 0.2 mm filter (Minisart, Sartorius, Gottingen, Germany). This eluate was then dried in vacuo and the sample redissolved into 1 ml 0.05 M acetic acid, this being the same as the original starting volume. This sample was then passed through a 10,000 MWCO centrifugal ultrafilter (Ultrafree-MC, Millipore, Sydney, Australia) to yield the sample used for bioassay analysis (cold extract). An aliquot of this ultrafiltrate was then dried in vacuo and redissolved in an equal volume of 0.1N HCl and subjected to 90 8C for 5 min (AOAC extract). Solid samples (crabs, blood cells, liver and gut contents pellet) were extracted with a slightly modified protocol to that recommended by the AOAC for PST extraction and
testing. Instead of adding 1 ml of extraction solvent to each gram of tissue, 5 ml of 0.1N HCl was added prior to homogenisation using a Heidolph tissue disruptor and heating the mixture at 90 8C for 5 min. The extract was clarified by centrifugation at 20,000g for 20 min (4 8C). Supernatant was decanted and passed through a 10,000 MWCO centrifugal ultrafilter to give the sample used for analysis (AOAC extract). Unheated extracts of these samples were also performed following the above procedure except that the solvent was 80% ethanol acidified to pH 2.0 with HCl, with the samples kept on ice or at 4 8C at all times (cold extract). 2.3. Radioreceptor assays for paralytic shellfish toxins The saxiphilin [3H]STX binding and voltage-gated sodium channel (Na channel) binding assays were conducted as previously described (Llewellyn et al., 2001a,b) with the exception of using 5 nM [3H]STX. Saxitoxin concentration equivalents (STXeq) was determined by back-calculation from calibration curves using standard STX after measuring [3H]STX inhibition by extracts at a series of dilutions as explained in detail elsewhere (Llewellyn et al., 2001a,b). Calibration curves generated with standard STX were ((100 2 F )/F )0.9 £ 4.4 nM for Na channel and ((100 2 F )/F )1.2 £ 6.3 nM for saxiphilin, where F ¼ %[3H]STX bound in the presence of extracts relative to controls, n and IC50 were the Hill slope and concentration of STX which causes 50% inhibition in the calibration curves, respectively. 2.4. Chromatographic analysis for paralytic shellfish toxins Selected samples were lyophilised and redissolved into 1 ml Milli-Q purified water to be then fractionated using a Sephadex G-15 column (1.5 £ 20 cm2, Amersham Pharmacia Biotech, Sydney, Australia) equilibrated in Milli-Q purified water. The column was eluted with 30 ml water, followed successively by 30 ml 0.005 M acetic acid, 30 ml 0.01 M acetic acid, 30 ml 0.025 M acetic acid, 30 ml 0.05 M acetic acid, and finally by 30 ml of 0.1 M acetic acid. Fractions (5– 10 ml) were collected, lyophilised and the residue resuspended into 0.05 M acetic acid whereupon they were assayed in the Na channel assay as described earlier for the detection of [3H]STX displacing activity. Active fractions were then subjected to standard HPLC analysis and compared to toxin standards (Llewellyn et al., 2001a,b; Oshima, 1995). HPLC toxin profiles were converted to absolute toxin values by multiplying the concentration of each toxin in an extract by its toxicity relative to STX (Oshima, 1995) and summing to obtain the total extract toxicity in STXeq. Toxins were identified by comparison of retention times and fluorescence emission maxima with standards, the disappearance of peaks by eliminating postcolumn oxidation and spiking experiments.
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
1465
Table 1 Quantitation of total PST load of extracts of crabs and human post-mortem samples using Na channel and saxiphilin radioreceptor assays Sample
Extract type
Na channel (mg STXeq/100 ml or 100 g tissue)
Saxiphilin (mg STXeq/100 ml or 100 g tissue)
Z. aeneus individual 1
Colda AOACb Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC Cold AOAC
76.7 ^ 14.2 461.5 ^ 92.7 24.2 ^ 1.0 289.6 ^ 12.9 12.1 ^ 2.2 15.8 ^ 2.6 0.9 ^ 0.4 0.9 ^ 0.3 Not detected Not detected 4.4 ^ 1.3 4.2 ^ 1.4 Not detected 10.3 ^ 1.0 423.4 ^ 119.7 577.1 ^ 58.9 448.4 ^ 83.1 385.4 ^ 92.9
331.2 ^ 52.8 291.6 ^ 6.2 111.8 ^ 10.9 346.7 ^ 83.8 10.0 ^ 2.7 10.1 ^ 3.4 4.9 ^ 1.8 4.0 ^ 0.1 Not detected Not detected 4.1 ^ 1.8 8.9 ^ 3.2 Not detected Not detected 260.8 ^ 48.6 257.5 ^ 40.2 224.1 ^ 14.8 376.2 ^ 37.8
Z. aeneus individual 2 Urine Liver Bile Blood plasma Blood cells Gut contents pellet Gut contents supernatant a b
Cold extraction method described in Section 2.2. Extracted by modified AOAC extraction method, which uses heat and acid.
3. Results 3.1. Preamble, case report and post-mortem In 1999, United Nations personnel were located in East Timor during a time of political instability and as part of their duties, they undertook investigations of suspected criminal cases. One of the authors (MJD) was consultant forensic pathologist and was asked to examine the body of a slightly built but well nourished 32 year old East Timorese male found dead on Behau Beach on 1 October 2000, after participating in a party. An initial suspicion that the deceased was murdered precipitated a full forensic examination. Police investigation revealed the victim had collected two crabs and another marine creature tentatively identified as being a member of the Class Holothuroidea (Phylum Echinodermata) at about 9.30 a.m. After cooking the animals on an open fire, part of one of the crabs and the holothurian were eaten. The victim rejoined the party, reportedly ‘ran around’ and drank two cans of alcoholic beer. Soon after, the victim vomited and lay down to rest at 11.00 a.m., stating he was ‘exhausted’. An hour later, it was discovered that he was dead. Nothing remarkable was observed upon external examination of the victim. Internal examination did not reveal any obvious disease. The victim had moderate pulmonary congestion and oedema and marked congestion of the meningeal vessels but no other abnormalities were observed, especially of the heart, coronary arteries and brain. No illicit drugs or medications were detected by routine toxicological analysis. Histological samples of all tissues were prepared and again revealed no
significant abnormalities apart from confirming non-specific congestion and oedema. The uneaten crab and the remains of the crab that was partly consumed were both identified by Dr Gary Poore, Curator of Crustacea, Department of Zoology, University of Melbourne as Z. aeneus, a crab species from the family Xanthidae previously implicated in fatal human intoxications. The absence of obvious pathological and histological abnormalities indicated that palytoxin did not contribute to the victim’s death. This toxin is hemorrhagic to mammals and mediates toxicity through significant effects on cardiovascular, kidney, gastrointestinal and respiratory systems (Wiles et al., 1974). This can result in congestion of organs and histological abnormalities are apparent in liver, kidney, heart, pancreatic and intestinal tissues (Terao et al., 1992). All remaining post-mortem samples were therefore analysed for toxins with the background knowledge that primarily PSTs, but on occasion, tetrodotoxin have been detected from this species of crab (Arakawa et al., 1994; Hwang et al., 1996; Llewellyn and Endean, 1989; Raj et al., 1983). 3.2. Screening and quantitation with radioreceptor assays Table 1 lists the PST concentrations (STXeq) calculated from Na channel and saxiphilin inhibition results from the various post-mortem extracts. STXeq values were calculated from the nanomolar values derived from calibration curves using the molecular weight of STX2þ of 301 and the sample dilution factors. Statistical comparison of the two datasets was undertaken using the Student’s t-test and by calculating the Spearman’s correlation coefficient, treating
1466
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
Table 2 Toxin profiles of Z. aeneus individual 2 and the victim’s urine and gut content supernatant samples calculated from HPLC chromatograms. ND signifies that the peak could not be positively identified as dcSTX Toxin profiles (mol%)
Z. aeneus individual 2 Gut content supernatant Urine
Total toxicity (mg STXeq/100 ml or 100 g tissue)
GTX2
GTX3
NEO
dcSTX
STX
45.5 5.5 0.1
17.3 10.2 0.3
0 2.6 9.6
0 ND 41.3
37.2 81.8 48.7
those samples in which toxin was not detected as zero. A two-tailed paired t-test generated a t-statistic of 0.92, which corresponds to a probability value of 0.37 (df ¼ 17). The Spearman’s correlation coefficient was 0.83 ( p , 0.0005). The values obtained by these two assay methods are therefore not statistically different and closely correlated. 3.3. Chromatography and HPLC confirmation of PSTs from samples HPLC analysis was performed on the cold acetic acid extracts that contained enough toxin for confident analysis (i.e. uneaten crab, stomach content and urine samples), where the original toxin profiles would be maintained (Fig. 1). To remove interfering fluorescent compounds, it was necessary to fractionate the samples using Na channel guided fractionation on a Sephadex G-15 column. The uneaten crab contained STX (37%) and the singly sulphated toxin epimers GTX2 (46%) and GTX3 (17%) (Table 2). No C-class toxins (i.e. doubly sulphated PSTs) were detected in any samples. The gut content supernatant was dominated by STX (82%), with lesser amounts of neoSTX (almost 3%) and GTX2 (6%) and GTX3 (10%). dcSTX could not be positively confirmed in this sample due to the presence of interfering fluorescent peaks. The toxin profile of the victim’s urine sample, however, comprised only 50% STX and much lower levels of GTX2 (0.1%) and GTX3 (0.3%). Compared to the gut contents, there was an increased proportion of the neoSTX (10%) and dcSTX was clearly resolved and prominent comprising 41% of the urine toxin profile. The total toxin concentration of the uneaten crab was 163 mg STXeq/100 g (Table 2), higher than the toxicity detected in each assay of the cold acetic acid extracts. The toxicity detected in the urine sample (15 mg STXeq/100 ml) agreed with both of the assays whereas HPLC detected a lower level of toxicity in the gut (30 mg STXeq/100 ml) compared with the assay techniques.
4. Discussion It is not surprising the victim perished after eating a Z. aeneus crab. Between 65 and 100% of Z. aeneus crabs
162.8 29.9 15.3
collected from Japan, Philippines, Australia and Fiji were toxic as measured by mouse lethality bioassay (Koyama et al., 1983; Llewellyn and Endean, 1989; Negri and Llewellyn, 1998; Raj et al., 1983; Yasumura et al., 1986). Further still, many of the reported toxicity values for these crabs exceeded the 80 mg STXeq/100 g tissue limit widely used by regulators to prohibit harvesting of molluscan shellfish for human consumption (Van Egmond et al., 1992). For example, by using the conversion factor of 1 mouse unit equalling 0.18 mg STX (Johnson and Mulberry, 1966), the proportion of crabs that contained more than 80 mg STXeq/100 g tissue ranged from 44% from Fijian specimens (Raj et al., 1983) to 100% in the Philippines and Japan (Koyama et al., 1983). The Na channel and saxiphilin receptor assays have been shown to closely correlate to mouse bioassay quantifications (Doucette et al., 1997; Llewellyn et al., 2001a,b). The fact that saxiphilin is insensitive to TTX whereas the Na channel is sensitive to both PSTs and TTX has been used to differentiate extracts containing significant amounts of TTX (Negri and Llewellyn, 1998). The close and statistically significant agreement between the two radioreceptor assays in Table 1 indicate that TTX is an insignificant contributor to the crab’s toxicity. Toxicity calculated from summing individual components from HPLC traces agreed well with the receptor assays. In the gut supernatant, however, a lower toxicity was reported by HPLC. Toxin peaks may have been masked by unremoved interfering compounds. It is also possible that some of the fluorescent peaks detected may have been undescribed PST analogues that contributed to the toxicity measured by bioassay. Small amounts of toxicity were detected in blood, liver and urine. This agrees with previous results where PST elimination in molluscan intoxicated humans was primarily via urination (Gessner et al., 1997; Montebruno, 1993). The presence in blood confirms the pathway by which the toxin is distributed about the body with some toxin being resident in the liver as has been observed before with Chilean molluscan PSP victims (Montebruno, 1993). The PST profile in the gut supernatant was dominated by STX whereas the urine contained only 50% STX. The increased proportion of neoSTX in the victim’s urine indicates that oxidation of the N1 group may be an important mechanism of elimination (Eq. (1)). The urine sample also
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
1467
Fig. 1. Several examples of HPLC analysis of PSTs from samples analysed in this study. Trace A, C and E are using conditions targeting gonyautoxins 1–5 with A being toxin standards, C being of the toxic fractions from crab #2 and E is of a toxic urine fraction. Traces B, D and F are of analyses targeting STX, dcSTX and neoSTX with B being toxin standards, trace D being of the toxic fractions from crab #2 and trace F is of the same toxic urine fraction as in E. Peaks labelled non-PSTs elute at similar retention times as standards but were shown not to be PSTs by their disappearance after eliminating post-column oxidation and by spiking experiments.
contained high proportions of dcSTX, indicating hydrolysis of the carbamoyl group of STX to form its decarbamoyl analogue (Eq. (2)). 1. Oxidation of N1 group to form the hydroxy analogue STX ! neoSTX
ð1Þ
2. Hydrolysis of carbamoyl group to form decarbamoyl analogue (Cembella et al., 1993; Oshima, 1996;
Sullivan et al., 1983) STX ! dcSTX
ð2Þ
The uneaten remains of the consumed crab weighed only 10 g. Less than half of the crab was eaten so no more than 10 g of tissue was ingested. Using the maximum toxicity determination, that for the gut content pellet that had undergone AOAC extract preparation, the victim could not have eaten more than 60 mg STXeq. Taking into
1468
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469
account the victim’s weight of approximately 50 kg, this gives a lethal oral dose of 1–2 mg STXeq/kg. This is less than lethal doses previously reported for PSP which have ranged from as low as 6 mg STXeq/kg (Shumway, 1995) to 4375 mg STXeq/ kg (Rodrigue et al., 1990). It is unlikely that significant levels of TTX were present in the samples, based upon the correlation between the PST specific saxiphilin assay and the PST and TTX sensitive Na channel assays, the amount of TTX present would add little to overall toxin burden consumed by the victim. It is worth noting that the crab flesh eaten in this case may have contained more toxicity than the value reported here for the whole crabs. For example the victim’s gut content had a higher relative toxicity than the remains of the meal itself. It has previously been reported that toxicity levels can vary greatly between different tissues of Z. aeneus. For example, again using the conversion factor of 1 mouse unit equalling 0.18 mg STX (Johnson and Mulberry, 1966), the muscle of the cephalothorax had 720 mg STXeq/100 g tissue while the muscle from the chelae had 108,000 mg STXeq/100 g tissue (Konosu et al., 1969). The range of countries where crab ingestion has killed people now includes East Timor. It is quite likely that crab poisonings have occurred previously in East Timor as anecdotal reports to United Nations field medical staff revealed that suicides were occasionally achieved by the intentional consumption of known toxic crabs, indicating a local knowledge of the potential toxicity of these crabs. The likelihood of victims receiving medical aid is low considering that the time until death after ingestion can be extraordinarily short. In this case, the victim was dead within several hours of eating the crabs, an interval between ingestion and death reported previously for victims of crab (Hashimoto et al., 1967; Mote et al., 1978) and molluscan induced PSP (Rodrigue et al., 1990). Bearing in mind the complexity of a meal and the variety of means by which they can be prepared, one cannot discount the possibility that other compounds within the victim’s meal may have promoted toxin uptake, a worrisome consideration for those with a public health interest in these naturally occurring toxins. The detection of PSTs associated with commercial seafood in temperate waters such as mussels and clams is routine; however, the recreational fishing of crabs in the tropics remains largely unregulated. This type of activity, coupled with a lack of scientific infrastructure in the tropics, means that the number of PST victims from crab poisoning is likely to be underestimated. Only greater public and practitioner awareness of potentially toxic species and symptoms and the development of rapid, cost effective testing kits will result in a decrease in PST related fatalities in this region.
References Alcala, A., Halstead, B., 1970. Human fatality due to ingestion of the crab Demania sp. in the Philippines. Clin. Toxicol. 3, 609–611.
Alcala, A., Alcala, L., Garth, J., Yasumura, D., Yasumoto, T., 1988. Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon 26, 105–107. Anderson, D.M., 1994. Red tides. Sci. Am. 271, 62 –68. Arakawa, O., Noguchi, T., Shida, Y., Onoue, Y., 1994. Occurrence of carbamoyl-N-hydroxy derivatives of saxitoxin and neosaxitoxin in a xanthid crab Zosimus aeneus. Toxicon 32, 175 –183. Cembella, A., Shumway, S., Lewis, N., 1993. Anatomical distribution and spatio-temporal variation in paralytic shellfish toxin composition in two bivalve species from the Gulf of Maine. J. Shellfish Res. 12, 389–403. Doucette, G., Logan, M., Ramsdell, J., Van Dolah, F., 1997. Development and preliminary validation of a microtiter platebased receptor binding assay for paralytic shellfish poisoning toxins. Toxicon 35, 625– 636. Gessner, B.D., Bell, P., Doucette, G.J., Moczydlowski, E., Poli, M.A., Van Dolah, F., Hall, S., 1997. Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks. Toxicon 35, 711 –722. Gonzales, R.B., Alcala, A.C., 1977. Fatalities from crab poisoning on Negros Island, Phillippines. Toxicon 15, 169–170. Halstead, B.W., Cox, K.W., 1973. A fatal case of poisoning by the red-spotted crab Carpilius maculatus (Linnaeus) in Mauritius. Proc. R. Soc. Arts Sci. Mauritius 4, 27 –30. Hashimoto, Y., Konosu, S., Yasumoto, T., Inoue, A., Noguchi, T., 1967. Occurrence of toxic crabs in Ryukyu and Amami Islands. Toxicon 5, 85–90. Hwang, D.F., Tsai, Y.H., Chai, T.J., Jeng, S.S., 1996. Occurrence of tetrodotoxin and paralytic shellfish poison in Taiwan crab Zosimus aeneus. Fish. Sci. 62, 500–501. Johnson, H., Mulberry, G., 1966. Paralytic shellfish poison: serological assay by passive haemagglutination and bentonite flocculations. Nature 211, 747 –748. Konosu, S., Inoue, A., Noguchi, T., Hashimoto, Y., 1969. A further examination on the toxicity of three species of xanthid crabs. Nippon Suisan Gakkaishi 35, 88– 92. Koyama, K., Noguchi, T., Uzu, A., Hashimoto, K., 1983. Individual, local, and size-dependent variations in toxicity of the xanthid crab Zosimus aeneus. Nippon Suisan Gakkaishi 49, 1273–1279. Lau, C., Tan, C., Li, Q., Ng, F., Yuen, R., Khoo, H., 1995. Bioactivity and mechanism of action of Lophozozymus pictor toxin. Toxicon 33, 901– 908. Llewellyn, L.E., 1997. Haemolymph protein in xanthid crabs: its selective binding of saxitoxin and possible role in toxin bioaccumulation. Mar. Biol. 128, 599 –606. Llewellyn, L.E., 2001. Human fatalities in Vanuatu after ingestion of a crab, Daira perlata. Med. J. Aust. 175, 343– 344. Llewellyn, L., Endean, R., 1989. Toxicity and paralytic shellfish toxin profiles of the xanthid crabs, Lophozozymus pictor and Zosimus aeneus, collected from some Australian coral reefs. Toxicon 27, 596–600. Llewellyn, L.E., Doyle, J., Negri, A., 1998. A high throughput, microtitre plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin. Anal. Biochem. 261, 51 –56. Llewellyn, L.E., Doyle, J., Jellett, J., Barrett, R., Alison, C., Bentz, C., Quilliam, M., 2001a. Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate
L.E. Llewellyn et al. / Toxicon 40 (2002) 1463–1469 saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay. Food Add. Contam. 18, 970 –980. Llewellyn, L.E., Negri, A.P., Doyle, J., Baker, P.D., Beltran, E.C., Neilan, B.A., 2001b. Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis. Env. Sci. Tech. 35, 1445–1451. Montebruno, D., 1993. Paralytic shellfish poisoning in Chile. Med. Sci. Law 33, 243–246. Mote, G.E., Halstead, B.W., Hashimoto, Y., 1978. Occurrence of toxic crabs in the Palau Islands. Clin. Toxicol. 3, 579–607. Negri, A., Llewellyn, L., 1998. Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia. Toxicon 36, 283 –298. Oshima, Y., 1995. Post-column derivatization HPLC methods for paralytic shellfish poisons. In: Hallegraeff, G., Anderson, D., Cembella, A. (Eds.), Manual on Harmful Marine Microalgae, Intergovernmental Oceanographic Commission Manuals and Guides, 33. UNESCO, Paris, pp. 81– 94. Oshima, Y., 1996. Chemical and enzymatic transformation of paralytic shellfish toxins in marine organisms. In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (Eds.), Harmuful and Toxic Algal Blooms, Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 475 –480. Powell, C.L., Doucette, G.J., 1999. A receptor binding assay for
1469
paralytic shellfish poisoning toxins: recent advances and applications. Nat. Toxins 7, 393–400. Raj, U., Haq, H., Oshima, Y., Yasumoto, T., 1983. The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands. Toxicon 21, 547– 551. Rodrigue, D., Etzel, R., Hall, S., de Porras, E., Velasquez, O., Tauxe, R., Kilbourne, E., Blake, P., 1990. Lethal paralytic shellfish poisoning in Guatemala. Am. J. Trop. Med. Hyg. 42, 267 –271. Shumway, S., 1995. Phycotoxin-related shellfish poisoning: bivalve molluscs are not the only vectors. Rev. Fish. Sci. 3, 1–31. Sullivan, J., Iwaoka, W., Liston, J., 1983. Enzymatic transformation of PSP toxins in the Littleneck Clam (Protothaca staminea ). Biochem. Biophys. Res. Commun. 114, 465 –472. Terao, K., Ito, E., Yasumoto, T., 1992. Light and electron microscopic observation of experimental palytoxin poisoning in mice. Bull. Soc. Pathol. Exot. 85, 494–496. Tsai, Y., Hwang, D., Chai, T., Jeng, S., 1995. Occurrence of tetrodotoxin and paralytic shellfish poison in the Taiwanese crab Lophozozymus pictor. Toxicon 33, 1669–1673. Van Egmond, H., Speijers, G., Van den Top, H., 1992. Current situation on worldwide regulations for marine phycotoxins. J. Nat. Toxins 1, 67– 85. Wiles, J., Vick, J., Christensen, M., 1974. Toxicological evaluation of palytoxin in several animal species. Toxicon 12, 427 –433. Yasumura, D., Oshima, Y., Yasumoto, T., Alcala, A., Alcala, L., 1986. Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric. Biol. Chem. 50, 593– 598.