- Email: [email protected]
Steroidal alkaloid toxicity to fish embryos
Toxicology Letters, 66 (1993) 1755181 0 1993 Elsevier Science Publishers B.V. All rights reserved 037%4274/93/$06.00
175
TOXLET 02841
Steroidal alkaloid toxicity to fish embryos
Lade11 Crawford” and Richard M. Kocanb “US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA (USA)
and bSchool of Fisheries HF-15. University of Washington, Seattle, WA (USA)
(Received 20 August 1992) (Accepted 15 October 1992) Key words: Rainbow trout; Steroidal alkaloid toxicity; Fish embryos
SUMMARY Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC,, EC,, and EC,, as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids a-chaconine, cc-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.
INTRODUCTION
Experiences in the past, such as with the widely used sedative thalidomide (after years of use, it proved to be a teratogen) and the more recent scare over the use of the pesticide Alar on apples, has increased our awareness of the need for rapid, simple and cost effective toxicological and biological evaluation techniques and procedures [l]. The use of traditional laboratory animal test models for such evaluations is now being questioned by a variety of pundits on the grounds that such testing subjects these animals to unnecessary pain, suffering and death. This debate has moved beyond considering whether or not these developing concerns are appropriate and well
Correspondence to: Lade11Crawford, Ph.D., US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA.
176
founded. Indeed, what seemed earlier to be philosophical musing has rapidly become the basis for public policy. There is some tacit agreement between the lay public and the research community; that is, more careful (caring) judicious use of laboratory animals under appropriate scrutiny should be manifested and alternative laboratory species (presumably, phylogenically lower) should be sought as well as substituting where possible in vitro methodologies. One of us (LC) has an abiding interest in the hepatic cytochrome P-450 monooxygenase xenobiotic metabolizing system and its ability to detoxify secondary plant metabolites that may present a threat to public safety [24]. A current project involves the study of the potential hazards of Solanum steroidal alkaloids (especially potatoes). These alkaloids are suspected of a broad range of effects on varying animal species including man; these effects include spina bifidas, embryo toxicity, teratogenicity, etc. [5-111. In an attempt to better understand these effects, with the ultimate goal of controlling or eliminating the toxicities, we have established a battery of in vivo and in vitro tests, while yet adopting sensitivity to the developing concerns about such laboratory procedures. One of us (RMK) [12] and other researchers [13-161 have proposed that fish embryos and cell cultures may be suitable models for biological and toxicological evalutions. Such ichthyologic systems are cost effective yet simple to establish and maintain. These systems readily yield information on the toxicity of a compound and can also provide an easy methodology (cell culture) to study metabolism. Therefore, this study will determine the LC,, and teratogenic effects of some steroidal alkaloids on selected fish embryos and evaluate their potential as model animals for the study of such compounds. MATERIALS
AND METHODS
The protocols followed for conducting these experiments with fish embryos have been described earlier [15,16]. Briefly, each of the six compounds (a-chaconine, c1solanine, solanidine, solasodine, jervine and repin) were dissolved in a solvent, dimethylsulfoxide (DMSO) or ethylene glycol (EG) which were used as a carrier for dilution of the compounds in water. Repin and jervine were generously supplied by Drs. Ken Stevens and William Gaffield, respectively. All other chemicals were purchased from the Sigma Company, St. Louis, MO. DMSO is used routinely in this laboratory (RMK) as the carrier of choice for carcinogens, mutagens and environmental extracts in experiments on trout and other salmonids, as well as in fish cell cultures. Concentrations at or below 1% DMSO have not caused any discernible effects on treated animals or cells. Nonetheless, a preliminary study was done prior to this experiment whereby medaka embryos were exposed to each carrier, DMSO and EG, to determine their effect on mortality and teratogenesis. No effects were found. Therefore, DMSO at a final concentration of 1% was chosen as the preferred solvent. Initially, multiple lo-fold concentrations were set up to establish a working range. A second set of exposures was then carried out to establish a dose response and EC,, for toxicity and teratogenicity observations for each compound. Embryos of two species
177
of fish were evaluated for their suitability as model systems for evaluating the aforementioned steroidal alkaloid compounds and the sesquiterpene lactone, repin. The latter equine neurotoxin was added as a on-going effort to find a suitable alternative animal model for toxicological-biological evaluations of this compound which, heretofore, seem only toxic to horses [18]. The Japanese rice fish (Oryzius lutipes) and the rainbow trout (Oncorhynchus mykiss) were exposed to each of the six compounds beginning on the day of fertilization and continuing until early embryogenesis (first tissue differentiation). This was identified by the first appearance of pigment in the eyes at about 14 days. At this stage the compounds were washed off the embryos and they were incubated in clean dechlorinated city water until hatching. During incubation, the embryos were examined daily for mortalities. These were counted, removed and placed into Stockard’s solution for fixation and later evaluation. After hatching, the larvae were examined daily for several weeks for mortalities. Just before ‘buttonup’ (i.e., yolk resorption) the larvae were fixed in 5% formalin and examined under a dissecting microscope for physical defects. The data were transformed to percent response vs. exposure concentration. This was graphed and used to estimate the EC,, for each compound. The real data were also entered into an EPA PROBIT ANALYSIS program and, where possible, evaluated for the statistical EC,, EC,, and EC,, as well as associated confidence limits (+ 95% confidence interval) [19]. RESULTS
AND DISCUSSION
All of the compounds were soluble over the range tested with the exception of solanidine and jervine which were soluble only below about 10 &ml. Medaka embryos were only affected by ol-chaconine and a-solanine (data not shown), where concentrations of 5 ,&ml and 25 pug/ml, respectively, caused 100% mortality. Surviving embryos were reared and observed for 2 months post hatching; none of the embryos showed any signs of physical defects. Figures l-5 show that rainbow trout embryos responded to four of the six compounds; solanidine and jervine (data not shown) did not show a significant increase in embryo mortality as related to concentration (dose response), that is, over the limited range of solubility. Relative toxicities of the compounds were as follows: a-chaconine > solasodine >> repin = a-solanine. The most toxic of the six compounds, a-chaconine, had an estimated LC,, of 4.2 ,@ml with significant toxicity at 3 puglrnl. It produced a good dose response from 1 through 6 pg/ml. Probit analysis yielded EC, = 2.3 (+ 0.6) ,&g/ml, EC,, = 3.6 (? 0.4) pug/ml and EC,, = 5.5 (? 1.3) ,ug/ ml. Estimated LC,, for solasodine, repin and a-solanine were 6.4, 17.0, and 17.0 ,&ml, respectively; EC, were 4.7 (? 2.9) 7.8 (? 3.7) and 12.4 (+ 1.9) pg/ml, respectively; EC,, were 7.6 (? 2.5) 19.6 (? 3.8) and 16.3 (? 1.1) ,@ml, respectively; ECg9 were 12.1 (rt 7.7) 49.0 (& 3.0) and 21.5 (k 3.7) ,ug/m 1, respectively. Interestingly, the toxicity curve for a-solanine was quite steep between 15-25 ,@ml, rising from 30 to 100% mortality over this mere 10 &ml concentration. Solanidine was only slightly soluble; however, the estimated LC,, based on embryo mortality up to a concentra-
178
alpha-CHACONINE
Fig.
1. Graphical
illustrations
of percent varying
rainbow
concentrations
(mg/L)
trout
embryo
mortality
resulting
from
exposure
to
(mg/i) of chaconine.
tion of 10 &ml was 20.2 pug/ml. Probit analysis could not be computed for this compound because of insufficient data (i.e., partial kills) due to its low solubility. Jervine was also relatively insoluble and no relationship between toxicity and concentration was found. Larval mortalities for 3 weeks post hatching were not significantly different from the control group for any of the compounds tested. Visual microscopic evaluation of the larvae which survived revealed no significant or systematic physical abnormalities relative to the control group. It is interesting that the same order of magnitude for the toxicity of ol-chaconine and a-solanine is reported for the LC,, of a frog embryo
,
8J
I
/
-y
=
I
-6.459 + 9.6361x R=
I
0.99576
z
0 0
N
*
(D
SOLASODINE
Fig. 2. Graphical
illustration
co
N
(mg/L)
of percent rainbow trout embryo mortality concentrations (mg/l) of solasodine.
resulting
from exposure
to varying
179
REPIN
(mg/L)
Fig. 3. Graphical illustration of percent rainbow trout embryo mortality resulting from exposure to varying concentrations (mg/l) of repin.
teratogenesis assay (FETAX) [20]. However, these two alkaloids produced some terata in the frog embryo, especially a-chaconine. These two glycoalkaloids are the major alkaloids found in domestic potatos (Solanum tuberosum) [20]. Both contain the same aglycone, solanidine, but are esterified to different trisaccharide side chains. Namely, branched his-d-r_-rhamnosyl-/3-n-glucopyranose @?-chacotriose) is the trisaccharide in ol-chaconine and a-solanine contains branched a-rhamnopyranosyl-/?-D-glucopyranosyl-a-galactopyranose (cx-solatriose). These secondary plant metabolites have been reported to be toxic to humans, including about 30 deaths [20]. Such alkaloids have been reported to behave in a similar manner to cardiac glycosides with respect to effects on the heart, that is, depression of the central nervous system with initial
::
/
z
::
: alpha-SOLANINE
::
:
E
(mg/L)
Fig. 4. Graphical illustration of percent rainbow trout embryo mortality resulting from varying concentrations (mg/l) of a-solanine.
0
ln
0
SOLANIDINE
,”
::
:
(mg/L)
Fig. 5. Graphical illustrations of percent rainbow trout embryo mortality resulting from varying concentrations (mg/l) of soianidine.
disappearance of EEG signals followed by cessation of respiration and terminal loss of ECG signals in unanesthetized rabbits [Zl]. Our results and those of other investigators such as Friedman et al. [20], suggest that the higher toxicity of a-chaconine vs. a-solanine is inescapably due to the difference in the esterified trisaccharide side chain since they both have the same aglycone. Is this difference in toxicity then due to differences in cellular solubility and/or stereo specificity? It should be possible to investigate the mechanism or toxicity in fish cell cultures using radiolabeled glycoalkaloids. Such systems provide undifferentiated as well as differentiated tissues which can be used to study the metabolism and site of action of these as well as various other compounds. Furthermore, it is possible to investigate vertical transmission of toxicity from adult fish to the embryos via eggs. Female and embryo metabolism could be distinguished because there is no direct connection between female and embryo as occurs in mammals. Any metabolites formed by the female which are lipid soluble would appear in the yolk material, and its effect separated from that occurring after direct exposure of the embryos. In conclusion, rainbow trout embryos may well serve as a good model for the toxicological-biological evaluation of steroidal alkaloids. REFERENCES Keeler, R.F. (1984) Mammalian teratogenicity of steroidal alkaloids. In: W.D. Nes, G. Fuller and L.S. Tsai (Eds.), Isopentenoids in Plants: Biochemistry and Function, Marcel Dekker. New York, pp. 531562. Crawford, L. (1983) Microsomal P-450 induction by some secondary products from thermal oxidation of dietary lipids: Epidermal hyperplasia, mutagenicity and cytochrome P-450 activities. Cancer Lett. 21, 211-217.
181
3 Crawford, L., McDonald, G.M. and Friedman, M. (1990) Composition of sicklepod (Cossiu obtusfolia) seeds. J. Agric. Food Chem. 38 (11). 2169-2175. 4 Crawford, L. and Friedman, M. (1990) The effects of low levels of dietary toxic weed seeds (jimson weed, Datum strumonium and sicklepod, Cassiu obrusifolia) on the relative size of rat liver and levels and function of cytochrome P-450. Toxicol. Lett. 54, 175-l 8 1. 5 Nevin, N.C. and Merrett, J.D. (1975) Potato avoidance during pregnancy in women with a previous infant with either anacephaly and/or spina bifida. Br. J. Prev. Sot. Med. 29, 111-l 15. 6 Sharma, R.P., Wilhite, C.C., Shupe, J.L. and Salunkhe, D.K. (1979) Acute toxicity and histopathological effects of certain glycoalkaloids and extracts of Alternariu sol& or phytophoru infestance in mice. Toxicol. Lett. 3, 349-355. ,7 Kyzlink, V., Mikova, K. and Jelinek, R. (1981) Tomatine, solanine and embryotoxicity of unripe tomatoes. Scientific papers of the Prague Institute of Chemical Technology, E51,69-81. 8 Gaffield, W. and Keeler, R.F. (1984) Structure and stereochemistry of steroidal amine teratogens. In: M. Friedman (Ed.), Nutritional and Toxicological Significance of Food Safety, Plenum Press, New York, pp. 241-251. 9 Renwick, J.H., Claringbold, D.B., Earthy, M.E., Few, J.D. and McLean, A.C.S. (1984) Neural tube defects produced in Syrian hamsters by potato glycoalkaloids. Teratology 30, 371-381. 10 Keeler, R.F. (1986) Teratology of steroidal alkaloids. In: S.W. Pelletier (Ed.), Alkaloids: Chemical and Biological Perspective, John Wiley and Sons, New York, pp. 389485. 11 Baker, D.C., Keeler, R.F. and Gaffield, W. (1988) Mechanism ofdeath in Syrian hamsters gavaged with potato sprout material. Toxicol. Pathol. 16, 333-339. 12 Kocan, R.M., Sabo, K.M. and Landolt, M.L. (1985) Cytotoxicity/Genotoxicity: The application ofcell culture techniques to the measurement of marine sediment pollution. Aquatic Toxicol. 6, 165-I 77. 13 Sinnhuber, R.O., Hendricks, J.D., Wales, J.H. and Putnam, G.B. (1977) Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. NY Acad. Sci. 298, 389408. 14 Bols, N.C., Boliska, S.A., Dixon, D.G., Hodson, P.V. and Kaiser, K.L.E. (1985) The use of fish cell cultures as an indication of contaminant toxicity to fish. Aquatic Toxicol. 6, 1477155. 15 Hawkins, WE., Overstreet, R.M. and Walker, W.W. (1988) Carcinogenicity tests with small fish species. Aquatic Toxicol. 11, 113-128. 16 Babich, H. and Borenfreund, E. (1991) Cytotoxicity and genotoxicity assays with cultured fish cells: A review. Toxicol. in Vitro 5( 1), 91-100. 17 McKim, J.M. (1985) Early life stage toxicity tests. in: M.R. Gary and J.R. Petrocelli (Eds.), Fundamentals of Aquatic Toxicology Methods and Applications, Hemisphere Publishing, New York, pp. 58-95. 18 Riopelle, R.J., Boegman, R.J., Little, P.B. and Stevens, K.L. (1990) Neurotoxicity of sesquiterpene lactones. In: L.F. James, R.F. Keeler, P.R. Cheeke, E.M. Baily Jr. and M.P. Hegerty (Eds.), Proceedings of the 3rd International Symposium on Poisonous Plants, Iowa State University Press, Ames, IA, in press. 19 The US Environmental Protection Agency: Biological Methods and Control Branch (1988). In: Users Guide for a Computer Program for Probit’s Analysis of Data From Acute and Short-Term Chronic Toxicity Tests With Aquatic Organism, Cincinnati, OH. 20 Friedman, M., Rayburn, J.R. and Bantle, J.A. (1991) Developmental toxicology of potato alkaloids in the frog embryo teratogenesis assay-Xenopus (FETAX). Fed. Chem. Toxicol. 29(8), 537-547. 21 Nishie, K., Gumbmann, M.R. and Keyl, A.C. (1977) Pharmacology of solanine. Toxicol. Appl. Pharmacol. 19, 81-92.
175
TOXLET 02841
Steroidal alkaloid toxicity to fish embryos
Lade11 Crawford” and Richard M. Kocanb “US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA (USA)
and bSchool of Fisheries HF-15. University of Washington, Seattle, WA (USA)
(Received 20 August 1992) (Accepted 15 October 1992) Key words: Rainbow trout; Steroidal alkaloid toxicity; Fish embryos
SUMMARY Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC,, EC,, and EC,, as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids a-chaconine, cc-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.
INTRODUCTION
Experiences in the past, such as with the widely used sedative thalidomide (after years of use, it proved to be a teratogen) and the more recent scare over the use of the pesticide Alar on apples, has increased our awareness of the need for rapid, simple and cost effective toxicological and biological evaluation techniques and procedures [l]. The use of traditional laboratory animal test models for such evaluations is now being questioned by a variety of pundits on the grounds that such testing subjects these animals to unnecessary pain, suffering and death. This debate has moved beyond considering whether or not these developing concerns are appropriate and well
Correspondence to: Lade11Crawford, Ph.D., US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA.
176
founded. Indeed, what seemed earlier to be philosophical musing has rapidly become the basis for public policy. There is some tacit agreement between the lay public and the research community; that is, more careful (caring) judicious use of laboratory animals under appropriate scrutiny should be manifested and alternative laboratory species (presumably, phylogenically lower) should be sought as well as substituting where possible in vitro methodologies. One of us (LC) has an abiding interest in the hepatic cytochrome P-450 monooxygenase xenobiotic metabolizing system and its ability to detoxify secondary plant metabolites that may present a threat to public safety [24]. A current project involves the study of the potential hazards of Solanum steroidal alkaloids (especially potatoes). These alkaloids are suspected of a broad range of effects on varying animal species including man; these effects include spina bifidas, embryo toxicity, teratogenicity, etc. [5-111. In an attempt to better understand these effects, with the ultimate goal of controlling or eliminating the toxicities, we have established a battery of in vivo and in vitro tests, while yet adopting sensitivity to the developing concerns about such laboratory procedures. One of us (RMK) [12] and other researchers [13-161 have proposed that fish embryos and cell cultures may be suitable models for biological and toxicological evalutions. Such ichthyologic systems are cost effective yet simple to establish and maintain. These systems readily yield information on the toxicity of a compound and can also provide an easy methodology (cell culture) to study metabolism. Therefore, this study will determine the LC,, and teratogenic effects of some steroidal alkaloids on selected fish embryos and evaluate their potential as model animals for the study of such compounds. MATERIALS
AND METHODS
The protocols followed for conducting these experiments with fish embryos have been described earlier [15,16]. Briefly, each of the six compounds (a-chaconine, c1solanine, solanidine, solasodine, jervine and repin) were dissolved in a solvent, dimethylsulfoxide (DMSO) or ethylene glycol (EG) which were used as a carrier for dilution of the compounds in water. Repin and jervine were generously supplied by Drs. Ken Stevens and William Gaffield, respectively. All other chemicals were purchased from the Sigma Company, St. Louis, MO. DMSO is used routinely in this laboratory (RMK) as the carrier of choice for carcinogens, mutagens and environmental extracts in experiments on trout and other salmonids, as well as in fish cell cultures. Concentrations at or below 1% DMSO have not caused any discernible effects on treated animals or cells. Nonetheless, a preliminary study was done prior to this experiment whereby medaka embryos were exposed to each carrier, DMSO and EG, to determine their effect on mortality and teratogenesis. No effects were found. Therefore, DMSO at a final concentration of 1% was chosen as the preferred solvent. Initially, multiple lo-fold concentrations were set up to establish a working range. A second set of exposures was then carried out to establish a dose response and EC,, for toxicity and teratogenicity observations for each compound. Embryos of two species
177
of fish were evaluated for their suitability as model systems for evaluating the aforementioned steroidal alkaloid compounds and the sesquiterpene lactone, repin. The latter equine neurotoxin was added as a on-going effort to find a suitable alternative animal model for toxicological-biological evaluations of this compound which, heretofore, seem only toxic to horses [18]. The Japanese rice fish (Oryzius lutipes) and the rainbow trout (Oncorhynchus mykiss) were exposed to each of the six compounds beginning on the day of fertilization and continuing until early embryogenesis (first tissue differentiation). This was identified by the first appearance of pigment in the eyes at about 14 days. At this stage the compounds were washed off the embryos and they were incubated in clean dechlorinated city water until hatching. During incubation, the embryos were examined daily for mortalities. These were counted, removed and placed into Stockard’s solution for fixation and later evaluation. After hatching, the larvae were examined daily for several weeks for mortalities. Just before ‘buttonup’ (i.e., yolk resorption) the larvae were fixed in 5% formalin and examined under a dissecting microscope for physical defects. The data were transformed to percent response vs. exposure concentration. This was graphed and used to estimate the EC,, for each compound. The real data were also entered into an EPA PROBIT ANALYSIS program and, where possible, evaluated for the statistical EC,, EC,, and EC,, as well as associated confidence limits (+ 95% confidence interval) [19]. RESULTS
AND DISCUSSION
All of the compounds were soluble over the range tested with the exception of solanidine and jervine which were soluble only below about 10 &ml. Medaka embryos were only affected by ol-chaconine and a-solanine (data not shown), where concentrations of 5 ,&ml and 25 pug/ml, respectively, caused 100% mortality. Surviving embryos were reared and observed for 2 months post hatching; none of the embryos showed any signs of physical defects. Figures l-5 show that rainbow trout embryos responded to four of the six compounds; solanidine and jervine (data not shown) did not show a significant increase in embryo mortality as related to concentration (dose response), that is, over the limited range of solubility. Relative toxicities of the compounds were as follows: a-chaconine > solasodine >> repin = a-solanine. The most toxic of the six compounds, a-chaconine, had an estimated LC,, of 4.2 ,@ml with significant toxicity at 3 puglrnl. It produced a good dose response from 1 through 6 pg/ml. Probit analysis yielded EC, = 2.3 (+ 0.6) ,&g/ml, EC,, = 3.6 (? 0.4) pug/ml and EC,, = 5.5 (? 1.3) ,ug/ ml. Estimated LC,, for solasodine, repin and a-solanine were 6.4, 17.0, and 17.0 ,&ml, respectively; EC, were 4.7 (? 2.9) 7.8 (? 3.7) and 12.4 (+ 1.9) pg/ml, respectively; EC,, were 7.6 (? 2.5) 19.6 (? 3.8) and 16.3 (? 1.1) ,@ml, respectively; ECg9 were 12.1 (rt 7.7) 49.0 (& 3.0) and 21.5 (k 3.7) ,ug/m 1, respectively. Interestingly, the toxicity curve for a-solanine was quite steep between 15-25 ,@ml, rising from 30 to 100% mortality over this mere 10 &ml concentration. Solanidine was only slightly soluble; however, the estimated LC,, based on embryo mortality up to a concentra-
178
alpha-CHACONINE
Fig.
1. Graphical
illustrations
of percent varying
rainbow
concentrations
(mg/L)
trout
embryo
mortality
resulting
from
exposure
to
(mg/i) of chaconine.
tion of 10 &ml was 20.2 pug/ml. Probit analysis could not be computed for this compound because of insufficient data (i.e., partial kills) due to its low solubility. Jervine was also relatively insoluble and no relationship between toxicity and concentration was found. Larval mortalities for 3 weeks post hatching were not significantly different from the control group for any of the compounds tested. Visual microscopic evaluation of the larvae which survived revealed no significant or systematic physical abnormalities relative to the control group. It is interesting that the same order of magnitude for the toxicity of ol-chaconine and a-solanine is reported for the LC,, of a frog embryo
,
8J
I
/
-y
=
I
-6.459 + 9.6361x R=
I
0.99576
z
0 0
N
*
(D
SOLASODINE
Fig. 2. Graphical
illustration
co
N
(mg/L)
of percent rainbow trout embryo mortality concentrations (mg/l) of solasodine.
resulting
from exposure
to varying
179
REPIN
(mg/L)
Fig. 3. Graphical illustration of percent rainbow trout embryo mortality resulting from exposure to varying concentrations (mg/l) of repin.
teratogenesis assay (FETAX) [20]. However, these two alkaloids produced some terata in the frog embryo, especially a-chaconine. These two glycoalkaloids are the major alkaloids found in domestic potatos (Solanum tuberosum) [20]. Both contain the same aglycone, solanidine, but are esterified to different trisaccharide side chains. Namely, branched his-d-r_-rhamnosyl-/3-n-glucopyranose @?-chacotriose) is the trisaccharide in ol-chaconine and a-solanine contains branched a-rhamnopyranosyl-/?-D-glucopyranosyl-a-galactopyranose (cx-solatriose). These secondary plant metabolites have been reported to be toxic to humans, including about 30 deaths [20]. Such alkaloids have been reported to behave in a similar manner to cardiac glycosides with respect to effects on the heart, that is, depression of the central nervous system with initial
::
/
z
::
: alpha-SOLANINE
::
:
E
(mg/L)
Fig. 4. Graphical illustration of percent rainbow trout embryo mortality resulting from varying concentrations (mg/l) of a-solanine.
0
ln
0
SOLANIDINE
,”
::
:
(mg/L)
Fig. 5. Graphical illustrations of percent rainbow trout embryo mortality resulting from varying concentrations (mg/l) of soianidine.
disappearance of EEG signals followed by cessation of respiration and terminal loss of ECG signals in unanesthetized rabbits [Zl]. Our results and those of other investigators such as Friedman et al. [20], suggest that the higher toxicity of a-chaconine vs. a-solanine is inescapably due to the difference in the esterified trisaccharide side chain since they both have the same aglycone. Is this difference in toxicity then due to differences in cellular solubility and/or stereo specificity? It should be possible to investigate the mechanism or toxicity in fish cell cultures using radiolabeled glycoalkaloids. Such systems provide undifferentiated as well as differentiated tissues which can be used to study the metabolism and site of action of these as well as various other compounds. Furthermore, it is possible to investigate vertical transmission of toxicity from adult fish to the embryos via eggs. Female and embryo metabolism could be distinguished because there is no direct connection between female and embryo as occurs in mammals. Any metabolites formed by the female which are lipid soluble would appear in the yolk material, and its effect separated from that occurring after direct exposure of the embryos. In conclusion, rainbow trout embryos may well serve as a good model for the toxicological-biological evaluation of steroidal alkaloids. REFERENCES Keeler, R.F. (1984) Mammalian teratogenicity of steroidal alkaloids. In: W.D. Nes, G. Fuller and L.S. Tsai (Eds.), Isopentenoids in Plants: Biochemistry and Function, Marcel Dekker. New York, pp. 531562. Crawford, L. (1983) Microsomal P-450 induction by some secondary products from thermal oxidation of dietary lipids: Epidermal hyperplasia, mutagenicity and cytochrome P-450 activities. Cancer Lett. 21, 211-217.
181
3 Crawford, L., McDonald, G.M. and Friedman, M. (1990) Composition of sicklepod (Cossiu obtusfolia) seeds. J. Agric. Food Chem. 38 (11). 2169-2175. 4 Crawford, L. and Friedman, M. (1990) The effects of low levels of dietary toxic weed seeds (jimson weed, Datum strumonium and sicklepod, Cassiu obrusifolia) on the relative size of rat liver and levels and function of cytochrome P-450. Toxicol. Lett. 54, 175-l 8 1. 5 Nevin, N.C. and Merrett, J.D. (1975) Potato avoidance during pregnancy in women with a previous infant with either anacephaly and/or spina bifida. Br. J. Prev. Sot. Med. 29, 111-l 15. 6 Sharma, R.P., Wilhite, C.C., Shupe, J.L. and Salunkhe, D.K. (1979) Acute toxicity and histopathological effects of certain glycoalkaloids and extracts of Alternariu sol& or phytophoru infestance in mice. Toxicol. Lett. 3, 349-355. ,7 Kyzlink, V., Mikova, K. and Jelinek, R. (1981) Tomatine, solanine and embryotoxicity of unripe tomatoes. Scientific papers of the Prague Institute of Chemical Technology, E51,69-81. 8 Gaffield, W. and Keeler, R.F. (1984) Structure and stereochemistry of steroidal amine teratogens. In: M. Friedman (Ed.), Nutritional and Toxicological Significance of Food Safety, Plenum Press, New York, pp. 241-251. 9 Renwick, J.H., Claringbold, D.B., Earthy, M.E., Few, J.D. and McLean, A.C.S. (1984) Neural tube defects produced in Syrian hamsters by potato glycoalkaloids. Teratology 30, 371-381. 10 Keeler, R.F. (1986) Teratology of steroidal alkaloids. In: S.W. Pelletier (Ed.), Alkaloids: Chemical and Biological Perspective, John Wiley and Sons, New York, pp. 389485. 11 Baker, D.C., Keeler, R.F. and Gaffield, W. (1988) Mechanism ofdeath in Syrian hamsters gavaged with potato sprout material. Toxicol. Pathol. 16, 333-339. 12 Kocan, R.M., Sabo, K.M. and Landolt, M.L. (1985) Cytotoxicity/Genotoxicity: The application ofcell culture techniques to the measurement of marine sediment pollution. Aquatic Toxicol. 6, 165-I 77. 13 Sinnhuber, R.O., Hendricks, J.D., Wales, J.H. and Putnam, G.B. (1977) Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. NY Acad. Sci. 298, 389408. 14 Bols, N.C., Boliska, S.A., Dixon, D.G., Hodson, P.V. and Kaiser, K.L.E. (1985) The use of fish cell cultures as an indication of contaminant toxicity to fish. Aquatic Toxicol. 6, 1477155. 15 Hawkins, WE., Overstreet, R.M. and Walker, W.W. (1988) Carcinogenicity tests with small fish species. Aquatic Toxicol. 11, 113-128. 16 Babich, H. and Borenfreund, E. (1991) Cytotoxicity and genotoxicity assays with cultured fish cells: A review. Toxicol. in Vitro 5( 1), 91-100. 17 McKim, J.M. (1985) Early life stage toxicity tests. in: M.R. Gary and J.R. Petrocelli (Eds.), Fundamentals of Aquatic Toxicology Methods and Applications, Hemisphere Publishing, New York, pp. 58-95. 18 Riopelle, R.J., Boegman, R.J., Little, P.B. and Stevens, K.L. (1990) Neurotoxicity of sesquiterpene lactones. In: L.F. James, R.F. Keeler, P.R. Cheeke, E.M. Baily Jr. and M.P. Hegerty (Eds.), Proceedings of the 3rd International Symposium on Poisonous Plants, Iowa State University Press, Ames, IA, in press. 19 The US Environmental Protection Agency: Biological Methods and Control Branch (1988). In: Users Guide for a Computer Program for Probit’s Analysis of Data From Acute and Short-Term Chronic Toxicity Tests With Aquatic Organism, Cincinnati, OH. 20 Friedman, M., Rayburn, J.R. and Bantle, J.A. (1991) Developmental toxicology of potato alkaloids in the frog embryo teratogenesis assay-Xenopus (FETAX). Fed. Chem. Toxicol. 29(8), 537-547. 21 Nishie, K., Gumbmann, M.R. and Keyl, A.C. (1977) Pharmacology of solanine. Toxicol. Appl. Pharmacol. 19, 81-92.