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Okadaic acid
Okadaic acid Maria Virginia Soldovieri Dept. of Health Science - University of Molise, Via De Santis - 86100 Campobasso, Italy
ã 2009 Elsevier Inc. All rights reserved.
Introduction Okadaic acid (OA) is a toxin that accumulates in bivalves and causes diarrhetic shellfish poisoning. OA was named from the marine sponge Halichondria okadai, from which OA was isolated for the first time. It has also been isolated from another marine sponge, H. malanodocia, as a cytotoxin. The real producer of OA is a marine dinoflagellate. OA is a potent inhibitor of specific subclasses of serine-threonine phosphatases, with PP2A showing the highest sensitivity, followed by PP1, and by PP2B (calcineurin); OA has no effect on PP2C Cohen (1989). In contrast, members of the Mg++-dependent protein phosphatase (PPM) and tyrosine phosphatases (PTPs) families are not affected by OA Klumpp and Krieglstein (2002).
Nomenclature Name of the Clinical Form Related Names Source: EMTREE Chemical Names CAS Number
9,10-Deepithio-9,10-didehydroacanthifolicin 78111-17-8
Basic Chemistry Chemical Structure Structure
Chemical Formula Properties Physical Properties Molecular Weight Solubility
C44H68O13
805.12 Soluble in chloroform, ethanol, methanol, acetone, ethyl acetate, and DMSO; not soluble in water.
1
2
Okadaic acid
Targets-Pharmacodynamics Serine-threonine phosphatases are divided in phosphoprotein phosphatase (PPP) and Mg++-dependent protein phosphatase (PPM) gene families. The PPP family includes the most abundant protein phosphatases—PP1, PP2A, and PP2B—as well as more recently cloned enzymes such as PP4 (also known as PPX), PP5, PP6 (a functional homolog of budding yeast Sit4), and PP7. These phosphatases were initially divided according to biochemical parameters: type-1 protein phosphatases (PP1) are inhibited by heat-stable inhibitor proteins and preferentially dephosphorylate the b-subunit of phosphorylase kinase. In contrast, type-2 protein phosphatases (PP2) are insensitive to these inhibitors and preferentially dephosphorylate the alpha subunit of phosphorylase kinase. The type-2 enzymes were further subdivided into spontaneously active protein phosphatase (PP2A), Ca++-dependent protein phosphatase (PP2B, also known as calcineurin), and Mg++dependent protein phosphatase (PP2C). Later on, cDNA cloning revealed that PP1, PP2A, and PP2B belong to the same gene family, whereas PP2C is structurally different. Five PP2C isoforms, together with the pyruvate dehydrogenase phosphatase, constitute the distinct gene family PPM Klumpp and Krieglstein (2002). PPs show differential sensitivity to blockade by okadaic acid (OA), with PP2A showing the highest sensitivity (IC50 0.1 nM), followed by PP1 (IC50 = 10–15 nM), and then by PP2B (IC50 = 5 mM); 10 mM OA has no effect on PP2C Cohen (1989). However, some of the less abundant protein phosphatases (PP4, PP5, and PP6) are inhibited by OA in the same nanomolar concentration range as PP1 and PP2A. In contrast, members of the PPM and tyrosine phosphatases (PTPs) families are not affected by OA Klumpp and Krieglstein (2002). The catalytic subunit of PP2A is among the most conserved enzymes in species ranging from yeasts to mammals. The PP2A core enzyme consists of a 36 kDa catalytic subunit, or C subunit, and a 65 kDa scaffolding protein, known as the A or PR65 subunit. The PP2A core enzyme associates with a variable regulatory subunit to form a PP2A holoenzyme. The recent report of the crystal structure of the PP2A core enzyme bound to OA at 2.6 A˚ resolution has revealed that the binding pocket for this toxin is located right above the two manganese atoms and the active site of PP2A Xing et al (2006). Although PP1 and PP2A share approximately 50% sequence identity, comparison of this structure with that of PP1 bound to OA Maynes et al (2001) allows rationalization for the strong toxin preference for PP2A. Although many residues of PP2A that specifically recognize OA are conserved in PP1, the hydrophobic cage in the catalytic subunit of PP2A that accommodates the hydrophobic end of OA is absent in PP1.
Target Name(s): Serine-threonine phosphatases (PP)
Therapeutics Among drugs affecting protein phosphorylation, the development of antitumor protein kinase inhibitors is most advanced. Cyclosporine, in association with its cellular binding protein cyclophilin, is a potent and specific inhibitor of the Ca++/calmodulin-dependent protein phosphatase PP2B. Inhibition of PP2B prevents dephosphorylation of an isoform of NFAT (nuclear factor of activated T-cells). As a result, this transcription factor cannot enter the nucleus, production of interleukin-2 is suppressed and T-cell proliferation is reduced. Cyclosporine is widely used clinically as an immunosuppressant Salton (2005).
Okadaic acid
The inhibition of PP1/PP2A by okadaic acidhas been linked to an enhancement of proliferation in most cellular systems; as a matter of fact, okadaic acidacts in vivo as a tumor promoter Suganuma et al (1988), Cohen et al (1990). Nevertheless, PPs are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases, and neurodegeneration Klumpp and Krieglstein (2002). Adverse Effects Okadaic acidis the main toxin produced by dinoflagellates which can accumulate in the hepatopancreas of mussels and cause diarrhetic shellfish poisoning in consumers.
Pre-Clinical Research Pharmacokinetics The distribution and excretion of okadaic acid (OA) administered orally (75, 150, and 250 mg/kg) to mice has been examined by immunostaining method Ito et al (2002). Five minutes after administration, OA was systemically distributed, being detected in the lung, liver, heart, kidney, and small and large intestine. Bleeding and edema in the lung were also found, and the distribution of OA coincided with these injuries. The detection of OA continued for 2 weeks in the liver and blood vessels. Excretion from kidney, cecum, and large intestine began even after 5 min of the administration, and the excretion from intestine continued for 4 weeks. When given orally at a single dose of 50 mg/kg, the toxin was found to concentrate in intestinal tissue and stomach. When the dose of OA was increased from 50 to 90 mg/kg, the concentrations of the toxin in the intestinal content and feces increased proportionally. A good correlation was found between an increase of OA in the intestinal tissue and the diarrhea in animals given 90 mg/kg orally Matias et al (1999). The same Authors also found that this marine toxin is able to cross the transplacental barrier Matias and Creppy (1996), with possible teratogenic consequences. In fact, OA has been shown to cause mortality, delayed growth and embryo malformation in the Frog Embryo Teratogenesis Assay Xenopus (FETAX). This is a screening assay using embryos at the gastrula stage of the anuran Xenopus laevis to identify substances that may pose a developmental hazard in humans Casarini et al (2007). In addition, the genotoxic potential of OA has also been recognized Le Hegarat et al (2006). Potency
Value Units Mouse DOSE 1
mg/kg/day
Prep. and Route Cell Organ/ of Line/ Tissue Admin. Type
Oral
Effects
Exp. End Point
Reference Comments
A short- Tubaro et al term (2004) toxicity study after 7 days oral daily
Okadaic acid induced diarrhea, body weight loss, reduced food consumption, and the
3
4
Okadaic acid administration
LD50 225
mg/kg
i.p.
Tubaro et al (2003)
Mouse LD50 204
mg/kg
Intraperitoneal
LD50 12
mg/kg
Administration onto the skin
Aune et al (2007) Fujiki et al (1988)
death of 2/5 mice after 5 days. Necroscopy and/or light microscopy analysis revealed toxic effects mainly at forestomach (ulceration and hyperplasia), liver, and indirectly leading to body weight loss in mice, as well as atrophic signs in the lymphoid organs and exocrine pancreas. Electron microscopy of heart tissue showed alterations of mitochondria and fibers in myocardiocytes, although no apoptotic change was recorded. Before death, okadaic acid-treated mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem.
TDLo (lowest toxic dose). Tumorigenic: equivocal tumorigenic agent by RTECS
Okadaic acid
LD50 1
mg /application
Rat or salmon EC50 20 nM
EC50 300
nM
Administration onto the skin
Fujiki et al (1991)
Salmon Apoptosis hepato- induction cytes
Rat Apoptosis hepato- induction cytes
(Registry of Toxic Effects of Chemical Substances) criteria From 1 week after initiation with a single application of 100 mg of 7,12-dimethyl benz[a] anthracene (DMBA), Okadaic acid (OA) was applied to the skin of the back of mice, twice a week. OA gave high percentages (86%) of tumor-bearing mice and high average numbers of tumors per mouse. These data indicated that a potent tumorpromoting activity could be achieved by OA.
Fladmark Overall, salmon et al hepatocytes (1998) appeared more sensitive than rat hepatocytes to the apoptogenic effects of marine toxins Fladmark et al (1998)
Other Information – Web Sites A PubChem Compound summary is available at http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6917781
5
6
Okadaic acid
http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178682985879.htm This site contains information from a press release on human toxicity and environmental exposure involving okadaic acid. For additional toxicological information: http://www2.siri.org/msds/tox/f/q0/q74.html
Journal Citations Le Hegarat, L., Jacquin, A.G., Bazin, E., Fessard, V., 2006. Genotoxicity of the marine toxin okadaic acid, in human Caco-2 cells and in mice gut cells. Environ. Toxicol., 21(1), 55–64. Cohen, P., Holmes, C.F., Tsukitani, Y., 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci., 15(3), 98–102. Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K., Yamada, K., Sugimura, T., 1988. Okadaic acid: an additional non-phorbol-12-tetradecanoate-13acetate-type tumor promoter. Proc. Natl. Acad. Sci. USA, 85(6), 1768–1771. Tubaro, A., Sosa, S., Carbonatto, M., Altinier, G., Vita, F., Melato, M., Satake, M., Yasumoto, T., 2003. Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon, 41, 783–792. Tubaro, A., Sosa, S., Altinier, G., Soranzo, M.R., Satake, M., Della Loggia, R., Yasumoto, T., 2004. Shortterm oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice. Toxicon, 43(4), 439–445. Fladmark, K.E., Serres, M.H., Larsen, N.L., Yasumoto, T., Aune, T., Døskeland, S.O., 1998. Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes. Toxicon, 36(8), 1101–1114. Klumpp, S., Krieglstein, J., 2002. Serine/threonine protein phosphatases in apoptosis. Curr. Opin. Pharmacol., 2(4), 458–462. Cohen, P., 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem., 58, 453–508. Xing, Y., Xu, Y., Chen, Y., Jeffrey, P.D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J.B., Yigong, Y., 2006. Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins. Cell, 127, 341–353. Maynes, J.T., Bateman, K.S., Cherney, M.M., Das, A.K., Luu, H.A., Holmes, C.F., James, M.N., 2001. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1. J. Biol. Chem., 276, 44078–44082. Ito, E., Yasumoto, T., Takai, A., Imanishi, S., Harada, K., 2002. Investigation of the distribution and excretion of okadaic acid in mice using immunostaining method. Toxicon, 40(2), 159–165. Matias, W.G., Traore, A., Creppy, E.E., 1999. Variations in the distribution of okadaic acid in organs and biological fluids of mice related to diarrhoeic syndrome. Hum. Exp. Toxicol., 18(5), 345–350. Matias, W.G., Creppy, E.E., 1996. Transplacental passage of [3H]-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography. Hum. Exp. Toxicol., 15(3), 226–230. Casarini, L., Franchini, A., Malagoli, D., Ottaviani, E., 2007. Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay. Toxicol. Lett., 169(2), 145–151. Salton, S.R., 2005. Protein Phosphatases. Sci. STKE, 273, tr8. Aune, T., Larsen, S., Aasen, J.A.B., Rehmann, N., Satake, M., Hess, P., 2007. Relative toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid, based on acute intraperitoneal toxicity in mice. Toxicon, 49(1), 1–7. Fujiki, H., Suganuma, M., Yoshizawa, S., Nishiwaki, S., Winyar, B., Sugimura, T., 1991. Mechanisms of action of okadaic acid class tumor promoters on mouse skin. Environ. Health Perspect., 93, 211–214. Fujiki, H., Suganuma, M., Suguri, H., Yoshizawa, S., Takagi, K., Uda, N., Wakamatsu, K., Yamada, K., Murata, M., Yasumoto, T., Sugimura, T., 1988. Diarrhetic Shellfish Toxin, Dinophysistoxin-1, Is a Potent Tumor Promoter on Mouse Skin. Jpn. J. Cancer Res., 79(10), 1089–1093.
ã 2009 Elsevier Inc. All rights reserved.
Introduction Okadaic acid (OA) is a toxin that accumulates in bivalves and causes diarrhetic shellfish poisoning. OA was named from the marine sponge Halichondria okadai, from which OA was isolated for the first time. It has also been isolated from another marine sponge, H. malanodocia, as a cytotoxin. The real producer of OA is a marine dinoflagellate. OA is a potent inhibitor of specific subclasses of serine-threonine phosphatases, with PP2A showing the highest sensitivity, followed by PP1, and by PP2B (calcineurin); OA has no effect on PP2C Cohen (1989). In contrast, members of the Mg++-dependent protein phosphatase (PPM) and tyrosine phosphatases (PTPs) families are not affected by OA Klumpp and Krieglstein (2002).
Nomenclature Name of the Clinical Form Related Names Source: EMTREE Chemical Names CAS Number
9,10-Deepithio-9,10-didehydroacanthifolicin 78111-17-8
Basic Chemistry Chemical Structure Structure
Chemical Formula Properties Physical Properties Molecular Weight Solubility
C44H68O13
805.12 Soluble in chloroform, ethanol, methanol, acetone, ethyl acetate, and DMSO; not soluble in water.
1
2
Okadaic acid
Targets-Pharmacodynamics Serine-threonine phosphatases are divided in phosphoprotein phosphatase (PPP) and Mg++-dependent protein phosphatase (PPM) gene families. The PPP family includes the most abundant protein phosphatases—PP1, PP2A, and PP2B—as well as more recently cloned enzymes such as PP4 (also known as PPX), PP5, PP6 (a functional homolog of budding yeast Sit4), and PP7. These phosphatases were initially divided according to biochemical parameters: type-1 protein phosphatases (PP1) are inhibited by heat-stable inhibitor proteins and preferentially dephosphorylate the b-subunit of phosphorylase kinase. In contrast, type-2 protein phosphatases (PP2) are insensitive to these inhibitors and preferentially dephosphorylate the alpha subunit of phosphorylase kinase. The type-2 enzymes were further subdivided into spontaneously active protein phosphatase (PP2A), Ca++-dependent protein phosphatase (PP2B, also known as calcineurin), and Mg++dependent protein phosphatase (PP2C). Later on, cDNA cloning revealed that PP1, PP2A, and PP2B belong to the same gene family, whereas PP2C is structurally different. Five PP2C isoforms, together with the pyruvate dehydrogenase phosphatase, constitute the distinct gene family PPM Klumpp and Krieglstein (2002). PPs show differential sensitivity to blockade by okadaic acid (OA), with PP2A showing the highest sensitivity (IC50 0.1 nM), followed by PP1 (IC50 = 10–15 nM), and then by PP2B (IC50 = 5 mM); 10 mM OA has no effect on PP2C Cohen (1989). However, some of the less abundant protein phosphatases (PP4, PP5, and PP6) are inhibited by OA in the same nanomolar concentration range as PP1 and PP2A. In contrast, members of the PPM and tyrosine phosphatases (PTPs) families are not affected by OA Klumpp and Krieglstein (2002). The catalytic subunit of PP2A is among the most conserved enzymes in species ranging from yeasts to mammals. The PP2A core enzyme consists of a 36 kDa catalytic subunit, or C subunit, and a 65 kDa scaffolding protein, known as the A or PR65 subunit. The PP2A core enzyme associates with a variable regulatory subunit to form a PP2A holoenzyme. The recent report of the crystal structure of the PP2A core enzyme bound to OA at 2.6 A˚ resolution has revealed that the binding pocket for this toxin is located right above the two manganese atoms and the active site of PP2A Xing et al (2006). Although PP1 and PP2A share approximately 50% sequence identity, comparison of this structure with that of PP1 bound to OA Maynes et al (2001) allows rationalization for the strong toxin preference for PP2A. Although many residues of PP2A that specifically recognize OA are conserved in PP1, the hydrophobic cage in the catalytic subunit of PP2A that accommodates the hydrophobic end of OA is absent in PP1.
Target Name(s): Serine-threonine phosphatases (PP)
Therapeutics Among drugs affecting protein phosphorylation, the development of antitumor protein kinase inhibitors is most advanced. Cyclosporine, in association with its cellular binding protein cyclophilin, is a potent and specific inhibitor of the Ca++/calmodulin-dependent protein phosphatase PP2B. Inhibition of PP2B prevents dephosphorylation of an isoform of NFAT (nuclear factor of activated T-cells). As a result, this transcription factor cannot enter the nucleus, production of interleukin-2 is suppressed and T-cell proliferation is reduced. Cyclosporine is widely used clinically as an immunosuppressant Salton (2005).
Okadaic acid
The inhibition of PP1/PP2A by okadaic acidhas been linked to an enhancement of proliferation in most cellular systems; as a matter of fact, okadaic acidacts in vivo as a tumor promoter Suganuma et al (1988), Cohen et al (1990). Nevertheless, PPs are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases, and neurodegeneration Klumpp and Krieglstein (2002). Adverse Effects Okadaic acidis the main toxin produced by dinoflagellates which can accumulate in the hepatopancreas of mussels and cause diarrhetic shellfish poisoning in consumers.
Pre-Clinical Research Pharmacokinetics The distribution and excretion of okadaic acid (OA) administered orally (75, 150, and 250 mg/kg) to mice has been examined by immunostaining method Ito et al (2002). Five minutes after administration, OA was systemically distributed, being detected in the lung, liver, heart, kidney, and small and large intestine. Bleeding and edema in the lung were also found, and the distribution of OA coincided with these injuries. The detection of OA continued for 2 weeks in the liver and blood vessels. Excretion from kidney, cecum, and large intestine began even after 5 min of the administration, and the excretion from intestine continued for 4 weeks. When given orally at a single dose of 50 mg/kg, the toxin was found to concentrate in intestinal tissue and stomach. When the dose of OA was increased from 50 to 90 mg/kg, the concentrations of the toxin in the intestinal content and feces increased proportionally. A good correlation was found between an increase of OA in the intestinal tissue and the diarrhea in animals given 90 mg/kg orally Matias et al (1999). The same Authors also found that this marine toxin is able to cross the transplacental barrier Matias and Creppy (1996), with possible teratogenic consequences. In fact, OA has been shown to cause mortality, delayed growth and embryo malformation in the Frog Embryo Teratogenesis Assay Xenopus (FETAX). This is a screening assay using embryos at the gastrula stage of the anuran Xenopus laevis to identify substances that may pose a developmental hazard in humans Casarini et al (2007). In addition, the genotoxic potential of OA has also been recognized Le Hegarat et al (2006). Potency
Value Units Mouse DOSE 1
mg/kg/day
Prep. and Route Cell Organ/ of Line/ Tissue Admin. Type
Oral
Effects
Exp. End Point
Reference Comments
A short- Tubaro et al term (2004) toxicity study after 7 days oral daily
Okadaic acid induced diarrhea, body weight loss, reduced food consumption, and the
3
4
Okadaic acid administration
LD50 225
mg/kg
i.p.
Tubaro et al (2003)
Mouse LD50 204
mg/kg
Intraperitoneal
LD50 12
mg/kg
Administration onto the skin
Aune et al (2007) Fujiki et al (1988)
death of 2/5 mice after 5 days. Necroscopy and/or light microscopy analysis revealed toxic effects mainly at forestomach (ulceration and hyperplasia), liver, and indirectly leading to body weight loss in mice, as well as atrophic signs in the lymphoid organs and exocrine pancreas. Electron microscopy of heart tissue showed alterations of mitochondria and fibers in myocardiocytes, although no apoptotic change was recorded. Before death, okadaic acid-treated mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem.
TDLo (lowest toxic dose). Tumorigenic: equivocal tumorigenic agent by RTECS
Okadaic acid
LD50 1
mg /application
Rat or salmon EC50 20 nM
EC50 300
nM
Administration onto the skin
Fujiki et al (1991)
Salmon Apoptosis hepato- induction cytes
Rat Apoptosis hepato- induction cytes
(Registry of Toxic Effects of Chemical Substances) criteria From 1 week after initiation with a single application of 100 mg of 7,12-dimethyl benz[a] anthracene (DMBA), Okadaic acid (OA) was applied to the skin of the back of mice, twice a week. OA gave high percentages (86%) of tumor-bearing mice and high average numbers of tumors per mouse. These data indicated that a potent tumorpromoting activity could be achieved by OA.
Fladmark Overall, salmon et al hepatocytes (1998) appeared more sensitive than rat hepatocytes to the apoptogenic effects of marine toxins Fladmark et al (1998)
Other Information – Web Sites A PubChem Compound summary is available at http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6917781
5
6
Okadaic acid
http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178682985879.htm This site contains information from a press release on human toxicity and environmental exposure involving okadaic acid. For additional toxicological information: http://www2.siri.org/msds/tox/f/q0/q74.html
Journal Citations Le Hegarat, L., Jacquin, A.G., Bazin, E., Fessard, V., 2006. Genotoxicity of the marine toxin okadaic acid, in human Caco-2 cells and in mice gut cells. Environ. Toxicol., 21(1), 55–64. Cohen, P., Holmes, C.F., Tsukitani, Y., 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci., 15(3), 98–102. Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K., Yamada, K., Sugimura, T., 1988. Okadaic acid: an additional non-phorbol-12-tetradecanoate-13acetate-type tumor promoter. Proc. Natl. Acad. Sci. USA, 85(6), 1768–1771. Tubaro, A., Sosa, S., Carbonatto, M., Altinier, G., Vita, F., Melato, M., Satake, M., Yasumoto, T., 2003. Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon, 41, 783–792. Tubaro, A., Sosa, S., Altinier, G., Soranzo, M.R., Satake, M., Della Loggia, R., Yasumoto, T., 2004. Shortterm oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice. Toxicon, 43(4), 439–445. Fladmark, K.E., Serres, M.H., Larsen, N.L., Yasumoto, T., Aune, T., Døskeland, S.O., 1998. Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes. Toxicon, 36(8), 1101–1114. Klumpp, S., Krieglstein, J., 2002. Serine/threonine protein phosphatases in apoptosis. Curr. Opin. Pharmacol., 2(4), 458–462. Cohen, P., 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem., 58, 453–508. Xing, Y., Xu, Y., Chen, Y., Jeffrey, P.D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J.B., Yigong, Y., 2006. Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins. Cell, 127, 341–353. Maynes, J.T., Bateman, K.S., Cherney, M.M., Das, A.K., Luu, H.A., Holmes, C.F., James, M.N., 2001. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1. J. Biol. Chem., 276, 44078–44082. Ito, E., Yasumoto, T., Takai, A., Imanishi, S., Harada, K., 2002. Investigation of the distribution and excretion of okadaic acid in mice using immunostaining method. Toxicon, 40(2), 159–165. Matias, W.G., Traore, A., Creppy, E.E., 1999. Variations in the distribution of okadaic acid in organs and biological fluids of mice related to diarrhoeic syndrome. Hum. Exp. Toxicol., 18(5), 345–350. Matias, W.G., Creppy, E.E., 1996. Transplacental passage of [3H]-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography. Hum. Exp. Toxicol., 15(3), 226–230. Casarini, L., Franchini, A., Malagoli, D., Ottaviani, E., 2007. Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay. Toxicol. Lett., 169(2), 145–151. Salton, S.R., 2005. Protein Phosphatases. Sci. STKE, 273, tr8. Aune, T., Larsen, S., Aasen, J.A.B., Rehmann, N., Satake, M., Hess, P., 2007. Relative toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid, based on acute intraperitoneal toxicity in mice. Toxicon, 49(1), 1–7. Fujiki, H., Suganuma, M., Yoshizawa, S., Nishiwaki, S., Winyar, B., Sugimura, T., 1991. Mechanisms of action of okadaic acid class tumor promoters on mouse skin. Environ. Health Perspect., 93, 211–214. Fujiki, H., Suganuma, M., Suguri, H., Yoshizawa, S., Takagi, K., Uda, N., Wakamatsu, K., Yamada, K., Murata, M., Yasumoto, T., Sugimura, T., 1988. Diarrhetic Shellfish Toxin, Dinophysistoxin-1, Is a Potent Tumor Promoter on Mouse Skin. Jpn. J. Cancer Res., 79(10), 1089–1093.