Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice

Toxicon 41 (2003) 783–792 www.elsevier.com/locate/toxicon

Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice A. Tubaroa,*, S. Sosaa, M. Carbonattob, G. Altiniera, F. Vitac, M. Melatod, M. Satakee, T. Yasumotof a DEMREP, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy LCG-RBM, Biomedical Institute of Research “A. Marxer” S.p.A., Colleretto Giacosa (TO), Italy c Department of Physiology and Pathology, University of Trieste, Trieste, Italy d Unit of Pathological Anatomy, University of Trieste, Trieste, Italy e Department of Applied Bioorganic Chemistry, Tohoku University, Sendai, Japan f Japan Food Research Laboratories, Tama Laboratories, Tokyo, Japan

b

Received 3 October 2002; accepted 11 February 2003

Abstract The acute toxicity of yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxy-homoyessotoxin (45-OH-homoYTX) has been studied in comparison to that of okadaic acid (OA), the main diarrhogenic toxin, both after intraperitoneal (i.p.) and oral administration. After i.p. administration, homoYTX and YTX showed similar lethality (LD50 ¼ 444 mg/kg and 512 mg/kg), higher than that of OA (LD50 ¼ 225 mg/kg), while 750 mg/kg of 45-OH-homoYTX did not cause death. OA induced the already known toxic signs: before death, mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem. Mice treated with YTX and homoYTX were restless and jumped before death; necroscopy did not show major changes. After oral treatment, 2 mg/kg of OA induced diarrhoea and body weight loss, causing 4/5 deaths; necroscopy and/or histology revealed degenerative lesions to small intestine, forestomach and liver (confirmed by increased plasma transaminase), but no myocardium alterations. On the contrary, the oral treatment with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not cause any death or signs of toxicity, except some ultrastructural myocardiocyte alterations, adjacent to capillaries, such as cytoplasmic protrusions (YTX, 1 and 2 mg/kg), fibrillar alteration (YTX, 1 mg/kg) or mitochondria assemblage (45-OH-homoYTX). Altogether, our data show that YTX and its derivatives are less toxic than OA after acute oral and i.p. treatments, at doses which may represent up to 100 times of the possible human daily intake. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Yessotoxin; Homoyessotoxin; 45-hydroxy-homoyessotoxin; Okadaic acid; Acute toxicity; Mice; Intraperitoneal administration; Oral administration

1. Introduction Yessotoxins (YTXs) are lipophilic polyether algal toxins with a structure similar to that of ciguatoxins and brevetoxins (Yasumoto and Murata, 1993). After the first isolation of YTX from the scallop Patinopecten yessoensis * Corresponding author. Tel.: þ39-040-558-7910; fax: þ 39-040558-3215. E-mail address: [email protected] (A. Tubaro).

in Japan (Murata et al., 1987), many YTX analogues have been isolated mainly in Europe, such as 45-hydroxy-YTX and 45,46,47-trinor-YTX (Satake et al., 1996), homoYTX and 45-hydroxy-homoYTX (Satake et al., 1997a), adriatoxin (Ciminiello et al., 1998), 1-desulfo-YTX (Daiguji et al., 1998), carboxy-YTX (Ciminiello et al., 2000a), carboxy-homoYTX (Ciminiello et al., 2000b) and 42,43,44,45,46,47,55-heptanor-41-oxo-homoYTX (Ciminiello et al., 2001). In particular, compounds of the YTX group are frequently detected in Mutsu Bay (Japan) and in

0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00032-1

784

A. Tubaro et al. / Toxicon 41 (2003) 783–792

Adriatic Sea (Italy) where, at present, they represent the main problem for the local shellfish industry. In fact, YTX was initially classified among the diarrhoetic toxins since it often coexists with okadaic acid (OA) and its derivatives, the major Diarrhoetic Shellfish (DS) toxins, and gives positive results when tested by the conventional mouse bioassay method for detecting DS toxins in shellfish (Ogino et al., 1997). Therefore, YTX shellfish contamination caused the prohibition of harvesting and commercialisation for several months, with severe economic losses for shellfish industries. However, the few existing studies showed that, unlike OA and its derivatives, YTX does not cause diarrhoea both in adult and in suckling mice (Murata et al., 1987; Ogino et al., 1997). The lack of diarrhogenic effects was also observed in rats treated with homoYTX-contaminated mussels (Tubaro et al., 1998). Furthermore, YTX is unable to affect the protein phosphatase 2A (Ogino et al., 1997), thus having a mechanism of action different from that of diarrhogenic toxins. As YTXs do not cause diarrhoea, recently the European Authorities set different limits for these compounds compared to other DS toxins (CEE, 2002). Anyway, the potential human toxicity of these compounds is not completely defined: only few toxicological data are available for YTX and none for its analogues. YTX seems to be much more toxic in mice after intraperitoneal (i.p.) injection (100 mg/kg) than after oral administration. The first data on acute toxicity after i.p. injection indicated YTX as the most toxic DS compound (Murata et al., 1987; Aune and Yndestad, 1993). Subsequent studies revealed conflicting values for LD50 after intraperitoneal injection. Terao et al. (1990), Ogino et al. (1997) and Towers (personal communication) recorded LD50 values ranging from 0.089 to 0.286 mg/kg, whereas the LD50 recorded by Aune et al. (2002) ranged between 0.75 and 1.0 mg/kg. The light microscopy analysis carried out by Terao et al. (1990) on mice treated with 0.3 mg/kg i.p. of YTX did not reveal any alteration in the main organs, whereas electron microscopy showed some changes in the heart tissue at 0.5 mg/kg. Aune et al. (2002) observed some heart tissue alterations at light microscopy, in mice treated i.p. with 1.0 and 0.75 mg/kg of YTX, but similar lesions were observed also in control animals. In the few available toxicity studies after acute YTX oral administration, none of the treated animals died, although dose levels ranging from 1.0 (Ogino et al., 1997) to 10 (Aune et al., 2002) or to 54 mg/kg (Towers, personal communication) have been administered to mice. Only at doses up to 7.5 mg/kg some changes in the behaviour of animals, which were slightly and transitorily affected, were observed (Aune et al., 2002). Slight intracellular myocardial oedema has been observed at light microscopy in animals treated with 7.5 and 10 mg/kg of YTX, although similar changes were noted also in a control animal. After oral administration of YTX (10 mg/kg), the electron microscopy

examination revealed changes in the myocardium similar to those induced by intraperitoneal injection of 1 mg/kg of the toxin (Aune et al., 2002). To better define the toxicological profile of YTX and its analogues homoYTX and 45-hydroxy-homoYTX, an acute toxicity study was carried out. Therefore, female CD-1 mice were given YTX, homoYTX and 45-hydroxy-homoYTX, either i.p. or per os. For comparison, similar doses of OA, the main diarrhogenic toxin, were given to other groups of mice.

2. Materials and methods 2.1. Toxins YTX was isolated from Protoceratium reticulatum collected in Mutsu Bay (Japan), according to the method of Satake et al. (1997a), while homoYTX and 45-hydroxyhomoYTX were isolated from Mytilus galloprovincialis collected in Adriatic Sea (Satake et al., 1997b). The purity of the final preparations, checked by NMR and MS spectroscopies (Yasumoto et al., 1995; Satake et al., 1997b), was more than 90%. OA (purity grade 98%) was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). 2.2. Animals and experimental conditions Female CD-1 mice, weighing 18 – 20 g, were purchased from Harlan Italy (S. Pietro al Natisone, Italy). Animals were kept for one week before the beginning of the experiments at controlled temperature (21 ^ 1 8C) and humidity (60 – 70%), using dust free poplar chips for bedding (Harlan Italy, S. Pietro al Natisone, Italy). Animals, fed with global diet for rodents containing 18.5% protein (Harlan Italy, S. Pietro al Natisone, Italy), were not fasted before the experiments. Toxins, dissolved in saline solution (0.9% NaCl) containing 1.8% ethanol (v/v), were administered to mice by intraperitoneal injection or per os, by gastric gavage, in a volume of 10 ml/kg body weight. Control animals received the vehicle alone (10 ml/kg of saline solution containing 1.8% ethanol v/v). Each dose of toxin or vehicle was administered to groups of at least three mice. All experiments were carried out in conformity with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC), concerning animal welfare. 2.3. Acute toxicity by intraperitoneal administration Three female mice were treated with each dose level of YTX (265, 375, 530 and 750 mg/kg), homoYTX (375, 530 and 750 mg/kg) and 45-hydroxy-homoYTX (750 mg/kg), while five or ten females were treated with each dose level

A. Tubaro et al. / Toxicon 41 (2003) 783–792

of OA (100, 159, 200, 252, 317 and 400 mg/kg). After intraperitoneal injection of the toxins, mice were observed for 24 h and signs of toxicity and mortality were recorded. Surviving animals were killed by ketamine hydrochloride (350 mg/kg i.p.) 24 h after the treatment. All the animals were weighed before the treatment and immediately after death. Each mouse was submitted to necroscopic examination and liver, heart, lungs, kidney and spleen were removed and weighed. Samples of these organs as well as of the stomach, duodenum, jejunum, colon, rectum, pancreas, thymus, uterus, ovaries, skeletal muscle, brain and spinal cord were fixed in 10% buffered formalin (0.1 M phosphate buffer, pH 7.0) for the histological analysis by light microscopy. Heart tissue was also analysed by terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling (TUNEL) immunostaining for the presence of apoptotic nuclei.

785

2.6. Light microscopy analysis and TUNEL staining of apoptotic nuclei Samples of the organs were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned (5 mm thickness). Sections were deparaffinized, rehydrated and stained with haematoxylin and eosin, following standard techniques, before being sent to LCG-RBM Biomedical Institute of Research “A. Marxer” S.p.A. who did the histological evaluation. Paraffin embedded heart tissue sections were submitted to the in situ detection of apoptotic nuclei by DNA fragmentation analysis. To this aim, DNA strand breaks were detected by terminal deoxynucleotidyl transferase-mediated BrdUTP nick end-labeling (TUNEL), using a commercially available kit (R&D Systems, Minneapolis, USA). 2.7. Transmission electron microscopy analysis

2.4. Acute toxicity by oral administration Groups of five female mice were orally treated with each dose level of YTX (1 and 2 mg/kg), homoYTX (1 mg/kg), 45-hydroxy-homoYTX (1 mg/kg) and OA (1 and 2 mg/kg). After oral administration, mice were observed for 24 h, recording signs of toxicity and mortality. Surviving animals were killed by ketamine hydrochloride (350 mg/kg i.p.) 24 h after the treatment. All the animals were weighed before the treatment and immediately after death. Blood samples from the abdominal aorta of all the animals were collected in heparinized syringes for the determination of the plasmatic levels of transaminases (aspartate-aminotransferase, AST; alanine-aminotransferase, ALT), lactate dehydrogenase (LDH), creatinine phosphokinase (CK), and for the percentages of the different leukocytes. Mice were submitted to necroscopic examination and the main organs were weighed and/or examined by light microscopy (see Section 2.3 for details). Moreover, heart tissue was analysed by TUNEL for the presence of apoptotic nuclei and, after fixing with glutaraldehyde, also by transmission electron microscopy. 2.5. Laboratory investigations (blood chemistry and haematology) Blood samples, obtained from orally treated animals, were analysed for the determination of plasmatic levels of AST, ALT, LDH and CK, which increase correlates with hepatic, cardiac or renal toxicity. These enzymes were measured by colorimetric methods using diagnostic kits obtained from Roche S.p.A. (Milan, Italy). A drop from each blood sample was smeared onto slides and stained with Giemsa stain, according to standard methodology. Neutrophils, eosinophils, basophils, lymphocytes and monocytes were counted on the basis of cell morphology and staining characteristics and their percentages were subsequently determined.

Heart tissue blocks were promptly fixed in a solution of 3% glutaraldehyde (Serva, Heidelberg, Germany) in 0.1 M cacodylate buffer, pH 7.3, for 3 h at 4 8C, rinsed three times (10 min each wash) in the same buffer and post fixed in 1% osmium tetroxide for 1 h at 4 8C. The samples were then dehydrated in a series of graded concentrations of ethanol and embedded in Dow Epoxy Resin (DER 332) (Lockwood, 1964). Ultrathin sections were cut by an ultratome Leica Ultracut UCT8 (Leica, Mikrosysteme Aktiengesellschaft, Wirn, Austria), double stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965) and examined with a Philips EM 208 transmission electron microscope. 2.8. Statistical analysis and determination of LD50 values The LD50 values (lethal dose of the toxins for 50% of the treated animals), based on 24 h mortality data, were calculated according to the method of Litchfield and Wilcoxon (1949) at the 95% confidence level. The significant differences between control and experimental groups were calculated using Student’s t-test, accepting p values lower than 0.05 as significant.

3. Results 3.1. Acute toxicity by intraperitoneal administration 3.1.1. LD50 values Mortality and symptoms of treated animals, reported in Table 1, show that death of mice injected with homoYTX or YTX occurred starting from the dose of 375 mg/kg. Their LD50 values (dose causing lethality in 50% of treated animals) were 444 and 512 mg/kg, respectively (95% confidence limits: 312– 618 and 315– 830 mg/kg for YTX and homoYTX, respectively). No mice lethality was

786

A. Tubaro et al. / Toxicon 41 (2003) 783–792

Table 1 Mortality induced by intraperitoneal injection of the toxins within 24 h Toxin

Dose (mg/kg)

Mortality

Survival times (h)

Symptoms

YTX

0 265 375 530 750

0/5 0/3 1/3 2/3 2/3

1.10 0.55–1.12 0.62–0.77

None None Restless, dyspnea, jumping Restless, dyspnea, jumping Restless, dyspnea, jumping

0 375 530 750

0/5 1/3 2/3 3/3

2.25 0.50–0.95 0.70–0.83–6.52

None Restless, dyspnea, jumping Restless, dyspnea, jumping Restless, dyspnea, jumping

45-OH-homoYTX

0 750

0/5 0/3

None None

OA

0 100 159 200 252 317 400

0/10 0/5 0/5 2/5 6/10 5/5 5/5

None Motionless Motionless Motionless, Motionless, Motionless, Motionless,

HomoYTX

recorded after administration of 750 mg/kg of 45-hydroxyhomoYTX. On the contrary, OA induced lethal effects starting from 200 mg/kg: its LD50 was 225 mg/kg (95% confidence limits: 176–275 mg/kg). 3.1.2. Symptoms Mice treated with homoYTX and YTX were restless and, in case of mortality, dyspnoea and jumping were observed before death. No symptoms were recorded for 45-hydroxyhomoYTX treated animals. On the contrary, mice treated with OA were motionless, cyanotic and presented dyspnoea before death (Table 1). 3.1.3. Necroscopic examination No significant differences in the final body and organ weights of mice in any of the administered compounds were seen. Necroscopic examination of YTX, homoYTX and 45hydroxy-homoYTX treated mice did not reveal particular signs of toxicity. The autoptic examination of mice treated with OA (200– 400 mg/kg) showed the presence of diffusely dark, or dark areas, in the liver. Duodenum and jejunum appeared hyperaemic and an accumulation of pale fluid was also noted.

3.48–11.27 7.75–5.83–8.73– 5.30–3.62– 1.95 5.28–2.52–3.17– 3.92–10.33 12.00– 2.90–2.62– 2.52–1.97

cyanosis, dyspnea cyanosis, dyspnea cyanosis, dyspnea cyanosis, dyspnea

On the contrary, histology of OA-treated mice revealed several morphological modifications of the intestinal tract, liver and myocardium. In particular, epithelium erosions, lamina propria congestion as well as shortening and flattening of villi were observed in duodenum and/or jejunum, with an incidence related with the dose of the administered toxin (Fig. 1). Moreover, all doses of OA induced moderate liver damage, consisting in isolated cell necrosis and/or vacuolisation of hepatocytes, which were dose-related both for their incidence and degree. Sporadically, hepatocellular changes were associated also with slight acute inflammation. Alterations of the myocardial tissue, i.e. slight-moderate haemorrhages, were observed in three of ten mice administered with doses of OA higher than 200 mg/kg. In order to verify whether the exposure to homoYTXs, YTX or OA induced apoptotic changes of myocardium, the in situ TUNEL staining of heart tissue for the presence of apoptotic DNA fragmentation was carried out. This procedure revealed no differences in the presence of apoptotic nuclei among controls and animals treated with YTX, its derivatives or OA. 3.2. Acute toxicity by oral administration

3.1.4. Light microscopy histologic examination Histologic examination of the main organs did not reveal morphological changes of tissues that could be clearly related to the treatments with YTX, homoYTX or 45hydroxy-homoYTX. Only one mouse treated with YTX (265 mg/kg, dose unrelated) and one treated with 45hydroxy-homoYTX (750 mg/kg) showed a limited haemorrhagic focus in the myocardial tissue.

3.2.1. Choice of the dose Orally administered doses of YTX and its analogues were determined considering the maximal human exposure to these toxins after mussel food consumption. In particular, since the highest concentration of YTX detected in mussels until now is 574 mg/100 g meat (Ramstad et al., 2001), a meal of such contaminated mussels (about 220 g) involves

A. Tubaro et al. / Toxicon 41 (2003) 783–792

787

Fig. 1. Light micrographs of duodenum (haematoxylin-eosin stain; objective magnification: 10 £ ). (a) Control: normal appearance of duodenum; (b) OA-treated mouse (252 mg/kg i.p.), showing atrophy of the villi, associated with scattered foci of epithelial erosion.

the consumption of 1263 mg of toxin. It means that, for a human of 70 kg, this amount corresponds to about 18 mg YTX/kg body weight, a dose that can be approximated to 20 mg/kg. Considering that in toxicological evaluation of environmental contaminants a safety factor of 100 is usually applied to extrapolate animal data to humans, the highest dose of YTX administered to animals in the present study was 2 mg/kg. Similarly, considering the maximal concentrations of homoYTX and 45-hydroxy-homoYTX till now detected in mussels (Tubaro et al., 1998; Ciminiello et al., 2001) the dose of 1 mg/kg of these toxins was estimated. Therefore, the maximal administered dose of YTX was 2 mg/kg, while homoYTX and 45-hydroxy-homoYTX were administered at 1 mg/kg. Likewise, the highest dose of OA was 2 mg/kg. Moreover, YTX and OA, the toxins available in the largest amount, were administered also at a lower dose (1 mg/kg), in order to better characterize the risk assessment. 3.2.2. LD50 values Oral administration of YTX (1 and 2 mg/kg) or its analogues (1 mg/kg) did not induce any lethal effect in

the treated mice. On the contrary, OA caused 4/5 deaths at the dose of 2 mg/kg, while no lethality was recorded at the lower dose (1 mg/kg), indicating an oral LD50 value between 1 and 2 mg/kg (Table 2). 3.2.3. Symptoms No symptoms of note were observed in mice orally treated with YTX (1 and 2 mg/kg) or its analogues (1 mg/kg). On the contrary, both the doses of OA (1 and 2 mg/kg) induced diarrhoea within 30 min after the administration. Moreover, mice treated with the lowest dose of OA (1 mg/kg) were motionless for about 1 h after the treatment, but subsequently they recovered. Also diarrhoea disappeared within 24 h. Similarly, mice treated with 2 mg/kg of OA were motionless, but subsequently they became hypothermic and cyanotic, presenting diarrhoea till death or final sacrifice (24 h). 3.2.4. Necroscopic examination No significant differences of the final body and organ weights between controls and YTXs treated mice were recorded. OA treated mice showed a reduction in body

Table 2 Mortality induced by oral administration of the toxins within 24 h Toxin

Dose (mg/kg)

Mortality

Survival times (h)

YTX

0 1 2

0/5 0/5 0/5

None None None

HomoYTX

0 1

0/5 0/5

None None

45-OH-homoYTX

0 1

0/5 0/5

None None

OA

0 1 2

0/5 0/5 4/5

None Diarrhoea, motionless, hypothermia, cyanosis Diarrhoea, motionless, hypothermia, cyanosis

6.87–8.40–9.15–21.00

Symptoms

788

A. Tubaro et al. / Toxicon 41 (2003) 783–792

Table 3 Effect of OA on plasma transaminases level Dose (mg/kg)

No. samples

AST (IU/l), m ^ SE

% increase

ALT (IU/l), m ^ SE

% increase

0 1 2

4 4 4

57.3 ^ 2.2 84.0 ^ 3.0a 425.3 ^ 122.6a

– 47 642

27.7 ^ 0.8 102.5 ^ 5.5a 201.7 ^ 38.3a

– 270 628

a

p , 0:05 at the Student’s t-test.

weight (6 – 7%, statistically significant at the dose of 2 mg/kg) and, at the dose of 1 mg/kg, also of the spleen weight (21%). Necroscopy of mice treated with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not show any macroscopic changes in the major organs. Anatomopathological examination of mice treated with OA (1 and 2 mg/kg) revealed dark areas on the liver and congestion of the stomach and small intestine. The latter was also distended and contained a pale or bloody fluid in its lumen. At the highest dose, an accumulation of pale fluid was observed also in the large intestine. 3.2.5. Light microscopy histologic examination Histology of the main organs and tissues of mice treated with YTX (1 and 2 mg/kg) or homoYTXs (1 mg/kg) did not show treatment-related morphological changes. On the contrary, one of five mice treated with 1 mg/kg and two of five mice treated with 2 mg/kg of OA showed degeneration of the duodenal villi, consisting in areas of moderate erosion of the epithelium, congestion of lamina propria and shortening of villi. The last change was observed also in the jejunum, without signs of erosion, but with intensely basophilic staining of the mucosa. The incidence of this change at the jejunum was dose-related, having been observed in one and five of five mice at the doses of 1 and 2 mg/kg, respectively. OA treatment (1 mg/kg) induced also some lesions at the forestomach of three of five mice, such as vacuolar degeneration of the epithelium, associated with acute inflammation of the submucosa. In addition, the forestomach of one mouse had reactive hyperplasia of the keratinized epithelium. On the contrary, at the highest dose of OA (2 mg/kg) no lesions of the gastric mucosa were observed. These animals showed slight spleen atrophy (depletion of the lymphoid elements), and degenerative modifications of hepatocytes were evidenced as slight-moderate cytoplasmic vacuolation.

To verify whether oral administration of homoYTXs, YTX or OA induced apoptotic damages on myocardium, the in situ TUNEL staining of heart tissue for apoptotic DNA fragmentation was carried out, observing no differences of apoptotic nuclei between controls and animals treated with YTX, with its derivatives or with OA.

3.2.6. Laboratory investigations (blood chemistry and haematology) The oral treatment with YTX (1 and 2 mg/kg), homoYTX (1 mg/kg) and 45-hydroxy-homoYTX (1 mg/kg) did not induce any significant difference in the plasmatic enzymes (AST, ALT, LDH and CK) as well as in the leukocytes percentages (data not shown). The oral administration of OA caused a dose-related, statistically significant increase of ALT (270 and 628% at the low and high doses, respectively) and AST (47 and 642%, respectively) (Table 3). The other monitored plasmatic enzymes (LDH and CK) as well as the leukocyte percentages were not affected by the toxin administration (data not shown).

3.2.7. Transmission electron microscopy analysis Electron microscopy analysis of heart tissue showed the presence of some alterations of myocardiocytes adjacent to capillaries in mice treated with YTX and its derivatives (Fig. 2). In particular, cytoplasmic protrusions of cardiac muscle cells into the pericapillary space, mitochondria rounded and packed and alterations of muscle fibres were observed in mice treated with both 1 and 2 mg/kg of YTX. Fibrillar structure alterations and a slight dilatation of some intercalated disks were noted only in mice treated with 1 mg/kg of YTX. Cytoplasmic protrusions of myocardiocytes, packed rounded mitochondria and fibres modifications were observed also in mice treated with homoYTX (1 mg/kg) and 45-hydroxy-homoYTX (1 mg/kg). Cardiac tissue analysis of OA treated mice was carried out only for the lower dose of the toxin (1 mg/kg) and no alterations

Fig. 2. Electron micrographs of myocardium (scale mark: 2 mm). (a) Control: normal appearance of muscle cells; (b) YTX (1 mg/kg): alteration of myocardiocyte visible as package of rounded mitochondria (large arrows) and changes of muscle fibers (small arrow); (c) homoYTX (1 mg/kg): rounded mitochondria (large arrows) and fibrillar alterations (small arrow); (d) homoYTX (1 mg/kg): particular of cell alteration near the capillary; (e) 45-hydroxy-homoYTX (1 mg/kg): general alterations of myocardiocyte, more pronounced near the capillary (mark); (f) OA (1 mg/kg): normal structure of myocardiocyte.

A. Tubaro et al. / Toxicon 41 (2003) 783–792

789

790

A. Tubaro et al. / Toxicon 41 (2003) 783–792

were observed, except some lipid droplets in myocardiocyte cytoplasm that were observed also in a control animal (Fig. 2).

4. Discussion There have been few previous studies on YTX toxicity. These studies report variable values for the toxin lethality in mice after intraperitoneal injection while no lethal effects are reported after its oral administration. Furthermore, no toxicological data exist on the analogues of YTX isolated until now. The present study was undertaken to give further contribution to the knowledge on the oral and intraperitoneal acute toxic effects in mice of homoYTX, 45hydroxy-homoYTX as well as of YTX. The toxic effects of these compounds were compared to those induced by the main DS toxin, okadaic acid. The results obtained in this study demonstrate that, after acute intraperitoneal administration, homoYTX (LD50 ¼ 444 mg/kg) and YTX (LD50 ¼ 512 mg/kg) possess similar lethal potencies and are more potent than 45hydroxy-homoYTX, which did not cause lethality at the dose of 750 mg/kg. Moreover, the lethal potency of YTX and of its two analogues was about two times lower than that of OA (LD50 ¼ 225 mg/kg). Therefore, the lethal potency of YTX observed in this study is in between the values observed by Terao et al. (1990), Ogino et al. (1997) and Towers (personal communication), with LD50s ranging from 0.089 to 0.286 mg/ kg, and those reported by Aune et al. (2002), with LD50s ranging between 0.75 and 1.0 mg/kg. These discrepancies can be related with variability of the experimental conditions between the laboratories and/or to different strains, age and sex of used mice. On the other hand, we find the same LD50 for OA as found by Tachibana and Scheuer (1981) and Dickey et al. (1990). Seventeen organs were histologically examined at light microscopy, and no alterations were observed after YTX (265, 375, 530 and 750 mg/kg), homoYTX (375, 530 and 750 mg/kg) and 45-hydroxy-homoYTX intraperitoneal administration (750 mg/kg). In particular, the moderate changes (intracellular vacuoles in myocardial muscle cells) observed by Aune et al. (2002) in the hearth of four of five mice which died after YTX treatment (0.75 and 1.0 mg/kg) were not recorded in any of the three mice treated with the highest YTX dose (750 mg/kg). Only a small hemorrhagic focus in myocardium has been observed in one mouse treated with YTX (265 mg/kg) and in one mouse treated with 45-hydroxy-homoYTX (750 mg/kg). However, being an isolated finding, in one case not related with the dose, it could not be clearly related to treatment effects. Furthermore, no apoptotic damages on the myocardium were detected by means of a specific immunostaining technique in mice treated either with YTX or with its analogues.

OA appeared to be more toxic than YTX or its analogues. After i.p. treatment, OA (200, 252 and 317 mg/kg) caused dose-related damages of the small intestinal mucosa. In particular, erosions of the duodenal and/or jejunum epithelium, lamina propria congestion as well as shortening and flattening of the intestinal villi were observed. In two of six animals (treated with 200 and 317 mg/kg) the villi, although shortened, did not show any erosion of the epithelium, but ongoing regenerative/reparative processes, as evidenced by the uniformly basophilic staining (hematoxylin and eosin) of the epithelium. These data confirm previous observations of damage of the epithelium of the upper small intestine, as well as on the reversibility of the intestinal damage (Ito and Terao, 1994). Moreover, for the first time, moderate myocardial hemorrhagic foci were observed in mice treated with 252 and 317 mg/kg of OA, even though not associated with apoptosis. OA i.p. treatment induced also moderate dosedependent hepatic degeneration (necrosis of isolated cells and/or vacuolation of hepatocytes), sometimes associated to acute inflammation. After per os acute administration, neither YTX (1 and 2 mg/kg) nor its analogues homoYTX (1 mg/kg) and 45hydroxy-homoYTX (1 mg/kg) induced lethality at the administered doses, which were equivalent to more than 100 times the human exposure after a large meal of highly contaminated mussels. On the other hand, the LD50 of OA orally administered ranged between 1 and 2 mg/kg. In our conditions, the oral toxicity of OA seems to be 5– 8 times lower than its i.p. toxicity. In particular, all the OA treated animals developed diarrhoea. As expected, diarrhoea was not observed in mice orally treated with the same doses of YTXs. The light microscopy histological examinations did not show any alteration of the 17 organs and/or tissues of animals treated with YTX, homoYTX and 45-hydroxyhomoYTX. However, electron microscopy of the myocardium revealed the presence of some alterations in mice treated with both YTX and its analogues. Cytoplasmic protrusions of cardiac muscle cells into pericapillary space and packing of rounded mitochondria were observed in mice treated with YTX (1 and 2 mg/kg), homoYTX (1 mg/kg) and 45-hydroxy-homoYTX (1 mg/kg), confirming previous observations in mice treated with YTX (10, 5 and 2.5 mg/kg p.o. or 0.5 mg/kg i.p.) (Aune et al., 2002). Fibrillar structure alteration was observed also in mice treated with YTX (1 mg/kg) and homoYTX (1 mg/kg). On the other hand, the oral administration of OA induced histological alterations to several organs. As after i.p. treatment, the main morphological changes were observed in the gastrointestinal system. However, regenerative/reparative processes were observed within 24 h, as previously reported (Ito and Terao, 1994; Ito et al., 2002). The oral OA treatment (1 mg/kg) caused forestomach erosions (three of five mice), in one case associated with reactive hyperplasia of the keratinized epithelium.

A. Tubaro et al. / Toxicon 41 (2003) 783–792

The same lesions were not observed at the highest dose of OA (2 mg/kg), probably because the animals died in a relatively short time. This is the first time that this kind of gastric lesions after oral OA administration are reported. Another toxicity target organ of OA is the liver: acute oral and intraperitoneal administration of OA provoked dosedependent hepatocyte degeneration, confirmed by a marked rise of the plasma transaminases levels. For the first time a liver damage is recorded after p.o. administration of OA. Recently, Ito et al. (2002) pointed out the lack of hepatotoxic effects of OA after oral administration, although microcystin-LR, sharing the same mechanism of action of OA, heavily affected the liver (Ito et al., 2000). Also Berven et al. (2001) did not find detectable effect on the liver after per os administration of OA (1 mg/g) in rats, but congestion of blood in the liver was observed after i.v. administration (0.2 mg/g). The necroscopic examination of the liver of OA-treated mice revealed alterations similar to those observable after microcystin-LR treatment (data not shown). The presence of liver changes in the present study is considered due to the high administered dose of OA (1 and 2 mg/kg), while the maximal dose previously administered to mice by oral route was 750 mg/kg (Terao et al., 1993; Ito and Terao, 1994). Furthermore, two of five mice showed slight spleen atrophy, probably consequent to the stress conditions of animals due to the treatment-induced direct toxicosis. After oral treatment, no changes were observed in the myocardium, where no apoptotic modifications were observed. Also the ultrastructural analysis did not reveal pathological modifications of the cardiac tissue of mice orally treated with OA (1 mg/kg). From the presented data, it can be concluded that the toxicological potential of YTX, homoYTX and 45-hydroxyhomoYTX is lower than that of OA, both after acute i.p. and oral administration. In particular, the damage of the small intestine is shown to be due to a systemic effect rather than to a local effect consequent to the oral route of administration. In fact, the same toxic effects were observed both after oral and parenteral route of administration. Difference seems to be in the mechanism of the toxic effect that OA induced in the non glandular gastric mucosa (forestomach) of mice. The i.p. treatment did not cause any gastric morphological alteration, while the acute oral treatment induces degeneration of the squamous epithelium and submucosal inflammation. Therefore, the observed gastric damage could be due to local irritation on the gastric mucosa, consequent to the oral administration of the toxin. However, it has to be taken into account that, while using these data for risk extrapolation to humans, the toxic effects on the forestomach, which is a tissue peculiar of rodents, are ‘species-specific’ and, consequently, not important and predictive for human toxicity. None of the three toxins of the YTXs group, administered at the same doses, provoked gastrointestinal or hepatic lesions. On the contrary, moderate ultrastructural changes of

791

the myocardial cells were detected both with YTX (1 and 2 mg/kg), homoYTX (1 mg/kg) and 45-hydroxy-homoYTX (1 mg/kg) orally administered. However, it has to be noted that no change has been observed in plasmatic LDH, an index of cardiac toxicity. Our results indicate that YTXs exert only moderate toxic effects on the myocardium, observable only by electron microscopy, in accordance with previous reports (Terao et al., 1990; Aune et al., 2002). However, from our results a Lowest Observable Effect Level (LOEL) of 1 mg/kg can be evaluated for YTX and homoYTX after oral exposure.

Acknowledgements This work was partially supported by ‘Regione FriuliVenezia Giulia’ and by a grant of the Italian Ministry of Instruction, University and Research (Project: ‘Tossine algali contaminanti i molluschi bivalvi nelle acque costiere italiane: caratteristiche, produzione, accumulo e azioni’). The authors are grateful to Mr Claudio Gamboz, ‘Centro Servizi Polivalenti di Ateneo’, University of Trieste, for his careful technical work. The authors are thankful also to Dr Michela Raffaele and Dr Claudia Casarsa for their skill full assistance, and to Dr Sergio Peano of LCG-RBM for revision of the manuscript.

References Aune, T., Yndestad, M., 1993. Diarrhetic shellfish poisoning. In: Falconer, I.R., (Ed.), Algal Toxins in Seafood and Drinking Water, Academic Press, London, pp. 87–102. Aune, T., Sorby, R., Yasumoto, T., Ramstad, H., Landsverk, T., 2002. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 40, 77–82. CEE, 2002. Commission Decision of 15 March 2002. Official J Eur Comunities. Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., Viviani, R., 1998. Isolation of adriatoxin, a new analogue of yessotoxin from mussels of the Adriatic Sea. Tetrahedron Lett. 39, 8897–8900. Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., Viviani, R., 2000a. A new analogue of yessotoxin, carboxyyessotoxin, isolated from Adriatic Sea mussels. Eur. J. Org. Chem. 2, 291 –295. Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., Viviani, R., 2000b. Structure determination of carboxyhomoyessotoxin, a new yessotoxin analogue isolated from Adriatic mussels. Chem. Res. Toxicol. 13, 770–774. Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., 2001. 42,43, 44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a new biotoxin from mussels of the Northern Adriatic Sea. Chem. Res. Toxicol. 14, 596– 599. Daiguji, M., Satake, M., Ramstad, H., Aune, T., Naoki, H., Yasumoto, T., 1998. Structure and fluorometric HPLC determination of 1-desulfoyessotoxin, a new analog isolated from mussels from Norway. Nat. Toxins 6, 235–239.

792

A. Tubaro et al. / Toxicon 41 (2003) 783–792

Dickey, R.W., Bobzin, S.C., Faulkner, D.J., Bencsath, F.A., Andrzejewski, D., 1990. Identification of okadaic acid from a Caribbean dinoflagellate. Prorocentrum concavum. Toxicon 28, 371–377. Ito, E., Terao, K., 1994. Injury and recovery process of intestine caused by okadaic acid and related compounds. Nat. Toxins 2, 371–377. Ito, E., Satake, M., Ofuji, K., Kurita, N., McMahon, T., James, K., Yasumoto, T., 2000. Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. Toxicon 38, 917 –930. 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, 159 –165. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96, 99–113. Lockwood, W.R., 1964. A reliable and easily sectioned epoxy embedding medium. Anat. Res. 150, 129–140. Murata, M., Kumagai, M., Lee, J.-S., Yasumoto, T., 1987. Isolation and structure of yessotoxin, a novel polyether compound implicated in diarrhetic shellfish poisoning. Tetrahedron Lett. 28, 5869–5872. Ogino, H., Kumagai, M., Yasumoto, T., 1997. Toxicologic evaluation of yessotoxin. Nat. Toxins 5, 255–259. Ramstad, H., Hovgaard, P., Yasumoto, T., Larsen, S., Anne, T., 2001. Monthly variations in diarrhetic toxins and yessotoxin in shellfish from coast to the inner part of Sognefjord, Norway. Toxicon 39, 1035–1043. Satake, M., Terasawa, K., Kadowaki, Y., Yasumoto, T., 1996. Relative configuration of yessotoxin and isolation of two new analogues from toxic scallops. Tetrahedron Lett. 37, 5955–5958.

Satake, M., MacKenzie, L., Yasumoto, T., 1997a. Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin. Nat. Toxins 5, 164 –167. Satake, M., Tubaro, A., Lee, J.-S., Yasumoto, T., 1997b. Two new analogs of yessotoxin, homoyessotoxin and 45-hydroxyhomoyessotoxin, isolated from mussels of the Adriatic Sea. Nat. Toxins 5, 107–110. Tachibana, K., Scheuer, P.J., 1981. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Halichondria. J. Am. Chem. Soc. 103, 2469–2471. Terao, K., Ito, E., Yanagi, T., Yasumoto, T., 1990. Histopathological studies on experimental marine toxin poisoning. 5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 28, 1095–1104. Terao, K., Ito, E., Ohkusu, M., Yasumoto, T., 1993. A comparative study of the effects of DSP-toxins on mice and rats. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea, Elsevier, Amsterdam, pp. 581 –586. Tubaro, A., Sidari, L., Della Loggia, R., Yasumoto, T., 1998. Occurrence of homoyessotoxin in phytoplankton and mussels from Northern Adriatic Sea. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Grafisant, Santiago de Compostela, pp. 470 – 472. Venable, J.H., Coggeshall, R., 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol. 25, 401–408. Yasumoto, T., Fujiki, M., Sasaki, K., Sugiyama, K., 1995. Determination of marine toxins in foods. J. AOAC Int 78, 574 –582. Yasumoto, T., Murata, M., 1993. Marine toxins. Chem. Rev. 93, 1897–1909.