Elsevier

Toxicon

Volume 57, Issue 5, April 2011, Pages 755-763
Toxicon

Acute oral toxicity in mice of a new palytoxin analog: 42-Hydroxy-palytoxin

https://doi.org/10.1016/j.toxicon.2011.02.009Get rights and content

Abstract

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300–1697 μg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD50 of 651 μg/kg (95% confidence limits: 384–1018 μg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 μg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥848 μg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424–1200 μg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure.

Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC50 = 7.6 and 13.2 × 10−12 M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na+/K+-ATPase as one of the key cellular targets of this toxin.

Introduction

Palytoxins (PLTXs) belong to a family of non-proteinaceous marine natural compounds, well known for their toxicity. Originally, parent PLTX was isolated in the Hawaiian Islands in 1971 from the cnidarian zoanthid Palythoa toxica (Moore and Scheuer, 1971). Since then, both PLTX and other molecules similar in structure, such as homopalytoxin, bishomopalytoxin, neopalytoxin, deoxypalytoxin, palytoxin-b and 42-hydroxy-palytoxin (42-OH-PLTX) have been identified in several tropical Palythoa species: P. vestitus (Wiles et al., 1974), P. tuberculosa (Uemura et al., 1985, Rossi et al., 2010), P. mammilosa, P. caribaeorum (Gleibs et al., 1995) and P. toxica (Ciminiello et al., 2009). In addition, PLTX or analogs have also been isolated from tropical benthic dinoflagellates of the genus Ostreopsis. Among them, the first was ostreocin-d (42-hydroxy-3,26-dimethyl-19,44-dideoxypalytoxin, OST-d), isolated from Ostreopsis siamensis (Ukena et al., 2001), later followed by mascarenotoxin-a, -b and -c, identified in Ostreopsis mascarenensis and Ostreopsis ovata (Lenoir et al., 2004, Rossi et al., 2010), and ovatoxin-a, -b, -c, -d and -e, identified in O. ovata (Ciminiello et al., 2006, Ciminiello et al., 2008, Ciminiello et al., 2010, Rossi et al., 2010).

PLTX and analogs have also been detected as seafood contaminants in crustaceans (Alcala et al., 1988, Yasumoto et al., 1986), fish (Onuma et al., 1999), and shellfish (Aligizaki et al., 2008, Aligizaki et al., 2010, Deeds and Schwartz, 2010, EFSA (European Food Safety Authority), 2009). To date, the presence of PLTXs in seafood has been directly related only to a few cases of lethal human poisoning in tropical areas (Alcala et al., 1988, Munday, 2008, Noguchi et al., 1987, Onuma et al., 1999, Taniyama et al., 2002, Tubaro et al., 2011). This phenomenon is becoming increasingly important due to PLTXs detection in shellfish and sea urchins in temperate regions (Aligizaki et al., 2008, Aligizaki et al., 2010, EFSA (European Food Safety Authority), 2009).

For a better comprehension of the effects of oral exposure to PLTX, in vivo toxicity studies were recently performed. These studies highlighted the relatively low toxicity of the compound after single oral administration in mice; lethality was recorded at doses approaching 1 mg/kg (Rhodes and Munday, 2004, Sosa et al., 2009). Alternative administration routes resulted in much higher potency: by intraperitoneal injection (i.p.), the toxin was lethal in mice at less than 1 μg/kg, with convulsions and breathing difficulties as primary signs before death (Riobò et al., 2008). Even though many in vitro and some in vivo observations documented the toxic effects of the parent PLTX, very few have dealt with the toxicity of its analogs. The major studies on PLTX analogs are related to OST-d. Acute i.p. administration to mice was lethal at 5 μg/kg, while oral administration (200 or 500 μg/kg) induced slight erosions in the stomach and small intestine, as well as toxic signs in the lungs and kidneys (Ito and Yasumoto, 2009).

Despite the claimed high human oral toxicity of PLTXs and their recent detection in edible marine organisms grown in the Mediterranean Sea, no regulatory limits have yet been set for these toxins. This may be due to the uncertainty of human epidemiological data (Tubaro et al., 2011) as well as to the few toxicological studies limited to PLTX and its analogue OST-d, especially after oral intake. The recent identification of a series of PLTX analogs (Ciminiello et al., 2010, Rossi et al., 2010) and the recent chemical characterization of 42-OH-PLTX, led us to study its acute oral toxicity in mice to better clarify the toxicological potential of this class of compounds.

The present toxicological study includes histological analysis of the main tissues and evaluation of some biochemical parameters indicative of cardiac, skeletal muscle, renal and/or hepatic damage, as well as the delayed hemolytic activity in mouse erythrocytes.

Section snippets

Toxins and other materials

Palytoxin, isolated from P. tuberculosa, was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan; lot number WKL7151, purity > 90%). Mouse monoclonal anti-PLTX antibody 73D3 was purified from culture medium by chromatography on a combination of Protein A and G Sepharose (Bignami et al., 1992). Other materials and chemicals were purchased from Sigma Aldrich (Milan, Italy).

Lethality

The incidence of death and the survival time of mice after 42-OH-PLTX oral administration are reported in Table 1. On the whole, the administration of 42-OH-PLTX (300–1697 μg/kg) was lethal in 16 out of 30 toxin-dosed animals, within 9 h post administration. The lowest lethal dose was 300 μg/kg inducing death in 1/5 mice and mortality reached 100% at the highest administered dose (1697 μg/kg). According to the method of Finney (1971), the calculated LD50 for 42-OH-PLTX was 651 μg/kg (95%

Discussion

From the reported results, after a single oral administration in mice of 42-OH-PLTX, the LD50 is calculated to be 651 μg/kg (95% confidence limits: 384–1018 μg/kg). This value is comparable to that of palytoxin determined in a previous study, where an LD50 of 767 μg/kg (95% confidence limits: 549–1039 μg/kg) was reported (Sosa et al., 2009). In the in vitro hemolysis test, the calculated EC50 for 42-OH-PLTX was comparable to that of PLTX: 7.6 × 10−12 and 13.2 × 10−12 M, respectively. The

Conclusions

42-OH-PLTX was the major toxic component of P. toxica collected from the Hana, Maui Hawaiian tidepool (Ciminiello et al., 2009), the source of palytoxin first reported in the ’70s (Moore and Scheuer, 1971). In this study, the toxicological effects of acute oral administration of 42-OH-PLTX were evaluated in mice, as well as its in vitro delayed hemolytic effects. The compound showed a toxicity profile comparable with that of the parent compound palytoxin (LD50 = 651 and 767 μg/kg,

Acknowledgments

This work was supported by the Italian Ministry of Education, University and Research (MIUR) [grant number 2007FXSCL2 005] and Friuli Venezia Giulia Region – Direzione Centrale Risorse Agricole, Naturali e Forestali. The authors thank Dr. Sergio Peano for the stimulating discussion.

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