Wayne T. Iwaoka

Enzymatic transformation of PSP toxins in the littleneck clam (Protothacastaminea)

Stemming from investigations into the relationship between toxins produced by Gonyaulax sp. and accumulated in shellfish, we wish to report enzymatic transformations of the PSP toxins to decarbamoyl derivatives in the littleneck clam (Protothaca staminea). No toxin transformations were observed in either mussels (Mytilus edulis) or in butter clams (Saxidomus giganteus). In addition, littleneck clam samples from the natural environment contained predominantly the decarbamoyl derivatives, while other shellfish species collected from the same vicinity contained the previously reported PSP toxins.

Benzo(a)pyrene-induced morphologic and developmental abnormalities in rainbow trout

To determine if the widespread environmental mutagen, benzo(a)pyrene (BaP), was embryotoxic or teratogenic in the rainbow trout (Salmo gairdneri R.), newly fertilized eggs were reared on sand experimentally coated with 1 to 500 ppm BaP. The system produced relatively constant aqueous BaP concentrations ranging from 0.08 to 2.99 ppb, levels comparable with those found in polluted rivers. Rainbow trout alevins reared in 2.40 ppb aqueous BaP for 36 days accumulated an average of 12.34 ppm BaP, and autoradiographic examination revealed accumulation of14C-benzo(a)pyrene primarily in the yolk sac and in developing neural and ocular tissues. Although no differences in either survival or hatching success were found between control and BaP-treated eggs, exposure to BaP did alter the length of the hatching process. Morphological abnormalities were significantly increased in BaP-treated alevins compared to controls at aqueous exposures of 0.21, 2.40, and 2.99 ppb. Insufficient yolk sacs, lack of body pigment, kyphosis, and abnormalities or absence of the eyes were among the anomalies present in alevins exposed to BaP.

Effects of benzo(a)pyrene on early development of flatfish

The ontogenetic effects of the environmental carcinogen benzo(a)pyrene (BAP) on three species of larval flatfish were investigated using concentrations (from 0.10 to 4.2 ppb) which were comparable to levels found in polluted harbors. BAP-treated sand sole (Psettichthys melanostichus) eggs displayed a significant decline in hatching success and a significantly higher incidence of developmental anomalies than did control eggs. Flathead sole (Hippoglossoides elassodon) eggs exposed to a single dose of a water-soluble BAP-bovine serum albumin complex demonstrated evidence of toxie injury with pycnotic nuclei present in the integument and, more commonly, in ocular and neural tissues. An increased incidence of morphological anomalies in English sole (Parophyrs vetulus) eggs and larvae exposed to BAP was not detected.

Linseed oil and animal fat as alternative lipid sources in dry diets for chinook salmon (Oncorhynchus tshawytscha)

Abstract Chinook salmon fingerlings (0.7 g) were fed experimental dry diets containing various levels of salmon oil, linseed oil and animal fat. The dietary levels of the fats were adjusted so that the essential fatty acid needs of the fish were met, but the source and the chain length varied with diet. No significant differences in final weight of the fish fed the experimental diets were observed after sixteen weeks of feeding. Moisture, protein, and lipid contents of the fish at the end of the experiment were the same. Analysis of the levels of fatty acids in the tissues of the fish showed that the fish maintain a constant level of saturated fatty acids regardless of the amount in the diet. The fatty acid composition of the neutral lipids generally reflected the fatty acid composition of the diets whereas the polar lipids selected for the more unsaturated fatty acids of the ω-3 series. Dietary linolenic acid (C18 : 3ω3) was not deposited in the body lipids as such but was apparently converted to docosahexaenoic acid (C22 : 6ω3).

Uptake of benzo[a]pyrene by gonadal tissue of flatfish (family Pleuronectidae) and its effects on subsequent egg development.

Accumulation of benzo[a]pyrene (BaP) by sexually mature flatfish gonad, its transfer to developing gametes, and its subsequent effects on developing embryos were studied. Thin-layer chromatography revealed both unmetabolized BaP and polar metabolites in the ovary, wolffian ducts, oocytes, and semen of English sole 24 h after ip injection with 200 microCi [3H]BaP. Concentrations of BaP and its metabolites were 3-11 times higher in oocytes and semen than in gonadal tissue. Fertilized eggs from flathead sole that had been fed 4.0 mg BaP 5 h before spawning demonstrated a significantly lower (p less than 0.001) hatching success (11.9%) than eggs from control fish (56.6%). Morphological abnormalities were found in only 1.6% of control embryos but in 5.6% of embryos from treated females.

Metabolism and lipogenic effects of the cyclic monomers of methyl linolenate in the rat

Cyclic fatty acids are absorbed by the rat, partially oxidized to CO2, and a portion of the compound, presumably the ring structure, is excreted in the urine. Studies with uniformly labeled cyclic fatty acids showed that ca. 13-15% of14CO2 is expired by the animal in 48 hr with peak expiration occurring between 4-6 hr after ingestion. Approximately 40% of the total radioactivity is found in the urine after 48 hr with about 60% of that being excreted within 12 hr after ingestion. Decreased rates of lipogenesis were observed in livers of animals fed 8% and 10% protein and higher levels of cyclic fatty acids. An increased rate of lipogenesis was observed in adipose tissue of animals fed 10% protein and higher levels of cyclic fatty acids.

Mutagen formation during the cooking of fish

Compounds mutagenic toward Salmonella typhimurium strains sensitive to frameshift mutation (1537, 1538 and TA98) were formed when fish flesh was fried at 190 degrees c. Four species of marine fish commonly consumed in the United States were cooked in an electric skillet and broiled beneath the elements of an electric oven. Organic extracts of the fish were tested in the Salmonella mutagenic assay using strains 1535, 1537, 1538, TA98 and TA100. Basic organic extracts of fried but not raw or broiled samples exhibited significant mutagenicity with metabolic activation. Mutagenic activity ratios ranging from 3.3 to 15.7 for the extract from 20 g of fish were observed. The mutagenicity produced during the frying of fish was dependent on time. Frying times of less than 6 min produced no mutagenic activity, while at 6 min or greater substantial mutagenicity was generated.

A rapid hemolysis assay for the detection of sodium channel-specific marine toxins

Current methods of detection for fish and shellfish biotoxins in monitoring and research purposes are either labor intensive, expensive, require specialized techniques or all of the above. This paper reports on the development of a fairly sensitive, rapid, and inexpensive assay which detects the presence of compounds that affect the sodium channel. It is based on the principles of the mouse neuroblastoma tissue culture assay for sodium channel specific-biotoxins using red blood cells (RBCs) from the red tilapia (Sarotherodon mossambicus). This assay has the potential to complement the use of live animal bioassay testing for marine toxins. Veratridine, a sodium channel activator and ouabain, an inhibitor of Na(+)/K(+) ATPase, both react with the tilapia RBCs by affecting the permeability of the cells membrane. Saxitoxin (STX), its analogs, and tetrodotoxin (TTX) can inhibit the action of veratridine and ouabain leaving the cell morphologically normal. By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected. Results using pure concentrations of a sodium channel-specific toxin could be detected to inhibit hemolysis at a concentration of 0.3 microg/ml STX, 3.5 microg/ml for neo-STX, 3.0 microg/ml for GTX, and 5.0 microgl for TTX in the presence of ouabain and veratridine. CTX was detected at a concentration of 50 microg/ml. The RBCs from the red tilapia was used due to the fishs ability to osmoregulate its internal environment to survive in both fresh and saltwater. In addition, with growing opposition to live animal testing, this assay has been designed as a non-lethal means of testing for sodium channel affecting marine toxins. No test animals are sacrificed and blood may be drawn from the same fish for continued sample testing.

A source of error in mutagen testing of foods

Mutagenic compounds reported to be present in foods may be forming during the extraction process rather than during cooking or baking. In this study, the formation of mutagenic substances in biscuits was examined using in the extraction procedure either ammonium sulfate and ammonium hydroxide or sodium sulfate and sodium hydroxide. Compounds producing high mutagenic activity in Salmonella strains 1538 and TA 98 obtained from aqueous biscuit extracts containing ammonium ions. No mutagenic activity was observed in extracts from aqueous biscuit extracts containing sodium ions until some ammonium ions (NH4OH) were added. We suggest that ammonium sulfate and ammonium hydroxide not be used in the extraction procedure of food when studying mutagen formation.

Effect of pH and ammonium ions on mutagenic activity in cooked beef

Controversy surrounding the extraction procedure commonly used for isolating and concentrating mutagens from foods has resulted in a need for the re-examination of the reported mutagenicity in fried hamburgers. Using a procedure in which Na2SO4 and NaOH are substituted for (NH4)2SO4 and NH4OH respectively, mutagenic activity in extracts of hamburgers fried for 5 min appeared to be unchanged. However, when organic extractions are performed at pH conditions more moderate than those generally employed to isolate mutagens from foods, a 30-50% decrease in mutagenicity is observed.