17
R e s p o n s e of t h e
House
Fly
to Saxitoxins
a n d C o n t a m i n a t e d Shellfish
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch017
ALVIN SIGER, BERNARD C. ABBOTT, and MARIA ROSS Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089
The problem of Paralytic Shellfish Poison as a hazard to human health and l i f e resulting from the consumption of bivalve marine molluscs that have ingested certain toxic dinoflagellates is well known. There is no known antidote and, although the majority of victims recover, there have been many fatalities when toxicity has been high. As a result the health authorities in the several coastal states require monitoring of shellfish and have set criteria for banning the collection of shellfish. The majority of shellfish are harvested by commercial organizations which regularly supply samples for analysis and as a result the number of incidents is kept remarkably low. However there exist long lengths of shoreline where native shellfish populations are harvested recreationally. The control imposed differs between States, varying from regular monitoring with the associated authority to ban collecting in defined local areas, to a State wide quarantine of a l l beaches for the collection of selected species during certain months of the year. The only legally recognized assay for PSP is that of the Mean Death Time of mice. Not only is this expensive but we believe that i t is very innacurate at low levels of toxicity. In addition, there are vast shellfish beds in remote areas of coastline that cannot be opened up for commercial explotation both because of the remoteness from mouse monitoring laboratories, and because of the expense. There is a recognized need for a alternative assay, but in reading the t i t l e of the communication a first reaction must be: "why a Bioassay?" and then "why a fly?". Preference is obviously for a simple chemical assay for PSP. Unfortunately the more specific the chemical test, the narrower is the window of compounds i t can assay. The Paralytic Shellfish Poison is not just Saxitoxin (STX) as originally believed, but is a mixture of compounds closely related to STX (1) and the mix varies widely with location and with time (2). It would seem, therefore that a chemical assay should determine at least the ratios of the several compounds, and that the relative toxicity of each of the compounds must be known. An effective assay must evaluate the actual biological toxicity of the shellfish being tested. For the chemical assay this requires the summated toxicity of a l l the 0097-6156/84/0262-0193$06.00/0 © 1984 American Chemical Society
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch017
194
SEAFOOD TOXINS
components and also assumes that there i s no synergism. Analysis and i d e n t i f i c a t i o n of the STX family of compounds i s essential i n the research studies on PSP but we believe that a bioassay giving the summed t o x i c i t y of samples w i l l s t i l l be needed for some time ahead. Why not the mouse? There seem to be several cogent objections to the Mouse Mean Death Time bioassay, although the sentiment i n support of a method that has been i n regular use for a quarter of a century i s very strong. Cost i s an ever present problem. In order to obtain consistent results the mice used i n any one laboratory must be of a defined s t r a i n , and of size 1 9 - 2 2 gr. wt Ç3). Such animals are expensive, and as more intensive monitoring i s demanded, costs are r i s i n g at a time when budgets are going down. The assay procedure i s to inject intrapertoneally 1 ml of the extract under test and observe the time to die. Unfortunately the death throeas are not very pleasant and i t has been c a l l e d the jump test. In an era of society's objection to the imposition of pain to mammals i n research and i n testing procedures, the government i s encouraging the development of alternatives. But of more concern i s what we f e e l to be an unacceptable error i n the monitoring of s h e l l f i s h that are only weakly toxic. This i s due to the " s a l t e f f e c t " observed by Schantz (4) and confirmed by McFarren (5) the following year. It refers to very considerable errors on the low side with weakly toxic s h e l l f i s h . In c a l i b r a t i n g the MTD method known concentrations of pure STX were added to tissue extracts of non-toxic clams, Schantz demonstrated f u l l recovery of the STX added down to the threshold l e v e l i f the samples were diluted with the low s a l t extraction buffer. I f , on the other hand, the d i l u t i o n was made with the (high s a l t ) non-toxic clam extract a progressive error was observed as d i l u t i o n increased, and near the threshold of detection the monitored l e v e l of STX was as much as 2 - 5 times too low. Thus for high levels of PSP the bioassay i s quite r e l i a b l e because the samples are diluted with the buffer, but for extracts that are less toxic and so need no d i l u t i o n , the assay i s made i n the presence of f u l l strength clam j u i c e and the error i s large. This can be very s i g n i f i c a n t for predictive e f f e c t s , which require the maximum s e n s i t i v i t y at the lowest levels of t o x i c i t y i n order to determine i f the early onset of a bloom may be detected from the pattern of low l e v e l s h e l l f i s h t o x i c i t y . The common house f l y Musca domestica i s paralyzed by STX i n a way that can be quantally scored ( 6 ) . This f l y i s ubiquitous. It can be purchased as pupae for a few cents or can be bred very simply i n any laboratory from wild stock for the price of a small volume of hamburger meat. The pupae can be stored for some time i f cooled and can be hatched at w i l l within a few days. The adult f l y emerges f u l l y grown and w i l l remain healthy for 2 0 - 3 0 days i f provided with water, sugar and dried milk. The method was i n i t i a l l y developed as an assay of purity of Leptinotarsin, a protein i n the hemolymph of the Colorado Potato Beetle (7) based on an idea of Fraenkel and Hsiao (8). Details of the method are given elsewhere (6). Enough f l i e s for an experiment are c h i l l e d and remain immobile i n a P e t r i dish on i c e . A volume of the solution being assayed, usually 1.5 y l i s injected into the f l y with a fine syringe needle. The f l y i s l e f t for 10 minutes at room
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch017
17.
SIGER E T AL.
Saxitoxins and Contaminated Shellfish
195
temperature and i s then quantally scored as Paralyzed or Active. At each d i l u t i o n of a sample, 10 f l i e s are injected and scored. The probit-log dose response r e l a t i o n i s l i n e a r f o r STX with a slope of 4.2 (±0.55) and an ED50 of 360 (+21, -19[S.D.]) picogram (for an i n j e c t i o n of 1.5 μΐ). This i s equivalent to toxin l e v e l i n a s h e l l f i s h of 48 yg/mg per 100 gr of s h e l l f i s h meat. Since, for the toxin and i t s associa ted assay protocol there appears to be a unique response i t i s possible to obtain an estimate of the median ED50 from one set of i n j e c t i o n s , provided that the dose i s not too far from the ED50 value. For STX t h i s represents a p r a c t i c a l l i m i t of detection of about 20 yg/100 g of meat. The f l y bioassay i s a simple, s e n s i t i v e and inexpensive method for determining the t o x i c i t y of a variety of neurotoxins. I t i s at least as s e n s i t i v e as the mouse Median Death Time for PSP and can be used as an alternative with the great advantage that i t does not display the s a l t e f f e c t . In fact the response of the f l y to toxic s h e l l f i s h extracts can be accounted for by assuming that there i s no " s a l t e f f e c t " as there i s i n the mouse Median Death Time method, but rather that "non-toxic" s h e l l f i s h contain a low l e v e l (approximately 10 yg/100 gm meat) of STX-equivalent material. This assumption permits a r e i n t e r p r e t a t i o n of the response of mice to toxic s h e l l f i s h and leads to agreement between the f l y r e s u l t s and those reported for the mouse on the same s h e l l f i s h samples. Combinatorial analysis gives the expected error d i s t r i b u t i o n s i n single dose-response estimations of a sample s toxin content. Chi-square analysis of 126 samples show that the observed error d i s t r i b u t i o n cannot be d i s t i n guished from those expected. 1
Literature Cited 1. 2.
3. 4. 5. 6. 7.
8.
Shimuzu, Y. In "Toxin Dinoflagellate Blooms," Taylor, D; Seliger, Η., Eds.; Elswert/North Holland; New York, 1979; p.321. Hall, S., Nave, R.A., Reichardt, P . S . , and Swisher, J . In "Toxic Dinoflagellate Blooms," Taylor, D; Seliger, H. Eds.; Elswert/North Holland; New York, 1979; p. 345. Official Method of Analysis. Assoc. Offic. Analyst. Chem. 1975, 38, 319. Schantz, E.J., McFarren, E . F . , Schafer, M . L . , Lewis, K.H. J . Assoc. Off. Agric. Chem. 1958, 41, 160. McFarren, E . J . J . Assoc. Off. Agric. Chem. 1959, 42, 263. Siger, Α . , Abbott, B . C . , Wong, T. Toxicon (in press). McClure, W.O., Abbott, B . C . , Baxter, D . E . , Hsiao, T . H . , Satin, L.S., Siger, Α . , Yoshino, J . E . Proc. Natl. Acad. Sci. USA 1980, 77, 1219-1223. Hsaio, T . H . , Frankel, G. Toxicon 1969, 7, 119-130.
RECEIVED July 20, 1984
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
