Toxicon 55 (2010) 87–91
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Effects of Madagascar yam extracts, Dioscorea antaly, on embryo-larval development of medaka fish, Oryzias latipes Lolona Rakotobe a, c, Miassa Berkal a, He´le`ne Huet b, Chakib Djediat a, Victor Jeannoda c, Bernard Bodo a, Lengo Mambu a, François Crespeau b, Marc Edery a, * a
FRE 3206 CNRS-USM 0505 Mole´cules de Communication et Adaptation des Micro-organismes, Muse´um National d’Histoire Naturelle, 12 rue Buffon, F-75231 Paris cedex 05, France b Laboratoire d’Anatomie Pathologique, Ecole Nationale Ve´te´rinaire d’Alfort, 7 Avenue du Ge´ne´ral de Gaulle, F-94704 Maisons-Alfort cedex, France c De´partement de Biochimie Fondamentale et Applique´e, Faculte´ des Sciences de l’Universite´ d’Antananarivo, B.P. 906, 101 Antananarivo, Madagascar
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 April 2009 Received in revised form 2 July 2009 Accepted 3 July 2009 Available online 10 July 2009
The yams edible starchy tubers, are of cultural, economic and nutritional importance in tropical and subtropical regions. The present study concerns the analysis at different levels of Dioscorea antaly toxicity to medaka embryo-larval development. The incubation of medaka fish embryos in a medium containing Dioscorea antaly extract resulted in a dose dependent reduction in survival rate. Survival rates were reduced up to 100% with extract concentrations of 4 mg mL1. The LD50 was estimated to be 0.86 mg mL1 Dioscorea antaly. Anatomopathological studies did not show any caustic effects, irritation to mouth, throat or intestinal tract in surviving embryos but rather an inflammatory reaction in the liver. The data presented in this paper thus extends the use of medaka embryos as a valuable model to analyze the effects of food toxins. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Yams Medaka Ecotoxicology Anatomopathology
1. Introduction The yams edible starchy tubers, are of cultural, economic and nutritional importance in tropical and subtropical regions (Coursey, 1967). In fact they are one of the main sources of food and nutrient energy for many people in the tropics. In Madagascar they represent an alternative supply of food to rice. The estimated yam production in the world is 40.106 tons (FAOSTAT, 2004). The yams belong to the Dioscoreaceae family. They are herbaceous plants with twine. Approximately 600 Dioscorea species are eaten in various parts of the world (Agbor-Egbe and Treche, 1995). The yams have been suggested to have higher nutritional properties when compared with other tropical root crops. They are reported as good sources of essential dietary nutrients (Splittstoesser et al.,1973; Baquar
* Corresponding author. Tel.: þ33 140793126; fax: þ33 140793594. E-mail address:
[email protected] (M. Edery). 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.07.002
and Oke, 1976; Egbe and Treche, 1984; Ologhobo, 1985; Bradbury, 1988; Wanasundera and Ravindran, 1994; Marcus et al., 1998; Cogne et al., 2001; Bhandari et al., 2003). However study reports have also pointed out that a few yam species contain some toxic compounds that can result in severe health problems (FAO,1990; Neuwinger,1994; Cogne et al., 2001). Some species of wild yams, particularly wild forms, are toxic and/or unpalatable, taste bitter and cause vomiting and diarrhea when large amount are ingested without proper processing or if eaten raw (Webster et al., 1984). A toxic principle in some yam species has been reported as dioscorine, a toxic alkaloid (David and Michael, 1985; Neuwinger, 1994; Webster et al., 1984). Dioscorine triggers the fatal paralysis of the nervous system when large fragments of the tuber weighing are ingested (Coursey, 1967). Similarly, histamine was reported as the principal allergen, causing mild inflammation and itching, associated with some plants of Dioscoreaceae family (Schmidt and Moult, 1983) although Dioscorea sylvatica was shown to induce mild inflammation at the skin level, mainly due to
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the presence of raphides of calcium oxalate (Cogne et al., 2001). On the other hand, the bitter substances in some yam species have been reported as furanoid-norditerpene groups of compounds (Kawasaki et al., 1968; Telek et al., 1974; Martin and Ruberte, 1976). The bitterness and toxicity of many yam species may also be caused by high level of saponins. Such is the case with Dioscorea tokoro in Japan or Dioscorea hispida in Thailand, where saponin is responsible for its bitterness (Tsukamoto and Ueno, 1936; Webster et al., 1984). In conclusion, the tubers with a high amount of protein, a good proportion of essential amino acids and in addition as a fairly good source of many dietary minerals provide a very advantageous nutrient (Bhandhari et al., 2003). However, their wider utilization is limited due to the presence of some toxic and antinutritional factors, if consumed without proper processing. Raw as well as cooked tubers are sometimes bitter, and the bitterness may be strong enough to render the tuber unpalatable. Ingestion of large amount of wild tubers has also been reported to cause caustic effects, irritation to mouth, throat or intestinal tract and absorptive poisoning. The local people prepare the tubers before consumption to make them palatable. The genus Dioscorea is distributed in tropical regions and in Madagascar, 32 species have been recorded and 27 of them are endemic such is the case of Dioscorea antaly. The tubers of Dioscorea antaly Jum. and H. Perrier (Dioscoreaceae) are used as food after prerequisite detoxification in water and dryness to remove bitters and toxic principles. Since no prior analyses of the toxic compounds of yam tubers have been carried out, the aim of the following study was to investigate on toxic principles in Madagascar yam tubers. Embryos of zebra and medaka fish are experimental tools in molecular developmental studies (Wittbrodt et al., 2002). They have also been used to analyse toxicity of toxins such as cyanotoxins on embryo-larval development (Jacquet et al., 2004; Huynh-Delerme et al., 2005; Escoffier et al., 2007; Lecoz et al., 2008). Small fish are becoming very popular for toxicity studies on many kinds of compounds as they allow to refine, reduce and replace other laboratory animals. The present study concerns the analysis at different levels of Dioscorea antaly toxicity to medaka embryo-larval development. The data presented in this paper thus extends the use of medaka embryos as a valuable model to analyse the effects of food toxins.
artificial reproductive regime (10 h light –14 h dark cycle). Fertilized egg clusters were carefully removed from the female progenitors and 650 post-fertilized (pf) eggs of medaka fishes were collected and separately gathered in Petri dishes containing Yamamoto’s embryo rearing medium (Yamamoto, 1975). Medaka embryos were then submitted to incubation experiments. For this purpose, Yamamoto’s embryo rearing medium was prepared with different extract concentrations. Embryos (5 in 2 mL) medium in 12 wells microplates were raised under the same conditions as described above (25 C, LD: 10–14 h) and observed daily and photographed (Leica) until 3 days post hatching (day 3 ph which corresponds to day 14 pf). Toxic media were changed during embryo development every 48 h. The animals were handled in accordance with European Union regulations concerning the protection of experimental animals (Dir. 86/609/EEC).
2. Materials and methods
Mortality rate (%)
88
2.1. Sample collection of tubers Dioscorea antaly tubers were collected on the wild in the Menabe region (Madagascar), essentially on May 2004. Voucher specimens (vouchers n MT 066, 067, 068 and 069) are deposited at the Botany Department of the Science Faculty of the Antananarivo University herbarium. Identification was done in the department. 2.2. Medaka breeding and treatment Medaka (Oryzias latipes) progenitors of the inbred cab strain were kept in 8-L glass aquaria at 26–28 C under
2.3. Anatomopathological studies At the end of the incubation experiments, surviving hatched embryos treated or not with extracts were fixed day 3 ph in buffered formaldehyde solution 4% (v/v). Fixed embryos were then clarified and dehydrated in three successive baths of ethanol (70, 95 and 100%) and finally kept in butanol (100%) overnight. Groups of 10 embryos were successively transferred into three baths of liquid
Survival rate (%)
A 100
controls 4mg/mL 2mg/mL 1mg/mL
80 60
0.5mg/mL 0.25mg/mL
40
0.125mg/mL 0.062mg/mL
*
20 0 0
B
1
2 Days post exposure
3
**
4
100 80 60 40 20 0 0.01
0.1
1
10
Dioscorea antaly extract (mg/mL) Fig. 1. (A) Survival rate of medaka embryos incubated with different concentrations of Dioscorea antaly ranging from 0.062 to 4 mg mL1. Bars represent mean SEM of 3–6 experiments. * denotes significant differences from controls (*p < 0.05, **p < 0.01). (B) Dose-response curve of mortality associated to Dioscorea antaly treatment. Bars represent the mean SEM of 3–6 experiments.
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Fig. 2. Light microscopy transverse sections of hatched (day 14 pf) control medaka embryos (A, C, E) and embryos treated with 0.5 mg mL1 of the Dioscorea antaly extract (B, D, F). (A, B) Mouth region, note the epithelial cells (arrows in A, B). (C, D) Kidney region, note the kidney tubules (arrows in C, D). (E, F) Liver, note the enlarged sinusoidal capillaries present in treated embryos (arrows in F) compared to controls (arrows in E). HE staining. Scale bars: 250 mm.
paraffin at 56 C and then oriented and embedded into blocks of paraffin wax. Transverse sections (3 mm thickness) were stained with haematoxylin-eosin (HES, Sigma, Aldrich, France). Microphotograph observations of histological transverse sections were carried out with an Olympus BX60 microscope at various magnifications.
between residual survival rates of each incubation series (control vs. treated embryos). A Tukey–Kramer’s multiple comparison test was then used to compare the responses of embryos in each dose range with that of controls.
3. Results and discussion 3.1. Preparation of Dioscorea antaly extracts
2.4. Statistical method analyses One factor ANOVA (Graphpad Prism 5 Software, http:// www.graphpad.com) was used to test the differences
Tubers were washed, cut as pieces of 0.5 cm, air-dried and milled. The powdered plant material was extracted with a large quantity of water at room temperature to
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avoid formation of a jelly past and concentrated immediately under reduced pressure. A rapid fermentation of the aqueous solution could be observed when stored at room temperature. The crude aqueous extract was conserved at 20 C.
The incubation of medaka fish embryos in a medium containing Dioscorea antaly extract resulted in a dose dependent reduction in survival rate. Dioscorea concentration of 4 mg mL1 reduced survival rate by 100% (Fig. 1A). Lower Dioscorea concentrations (2 and 1 mg mL1) resulted in higher survival rates (z20–30%). The lowest Dioscorea concentrations (0.5, 0.25, 0.1250, 0.062 and 0.031 mg mL1) were ineffective (90% survival, Fig. 1A). The LD50 was estimated to be 0.86 mg mL1 Dioscorea (Fig. 1B). No retardation of embryo development compared to the normal development as described by Yamamoto (1975) could be observed.
well as microalgae in sea water resources (Escoffier et al., 2007; Lecoz et al., 2008). They permit to analyse the toxicity of one of the main sources of food and nutrient energy for many people in the tropics; namely yams. Dose-response curves can be obtained indicating toxicity levels for an aquatic animal. The present anatomopathological studies did not show any caustic effects, irritation to mouth, throat or intestinal tract in surviving embryos but rather an inflammatory reaction in the liver similarly to what was reported recently in rats fed with Dioscorea villosa (Wojcikowski et al., 2008) however we did not notice any kidney alterations such as fibrosis as was observed in rats. Thus medaka embryos, which are widely used to monitor toxicity, could bring additional information on food safety associated with yam consummation and its relationship to environmental biohazard conditions. In addition target organs are detectable that can lead to specific research on the toxic effects. Two research domains are now explored in embryos and also adults: (1) the active molecules involved in the toxicity of the extracts; and (2) the induced proteomic modulations are studied.
3.3. Anatomopathological effects of Dioscorea antaly extracts on medaka embryo
Acknowledgments
3.2. Dioscorea antaly effects on medaka embryo-larval development
Anatomopathological studies were conducted on control embryos and embryos treated with a concentration of 0.5 mg/ml extract corresponding to the LD50. In Fig. 2, transverse sections of embryos in three different regions are shown; mouth, hepatic and kidney regions. There is no evidence of pathological modifications or irritation in the kidney, mouth regions or intestinal tract. In the liver, the general structures are conserved between controls and treated embryos with an enlargement of sinusoidal capillaries in treated embryos. Indeed, following Dioscorea treatment, there is a statistically significant increase (p < 0.001) in the width of these capillaries (Table 1) in the treated embryos, compared to the controls; similarly following treatment, the thickness of the capillaries is significantly increased (p < 0.001) (Table 1) in the treated embryos, compared to controls. This type of modification is suggestive of an inflammatory reaction in the liver, as a matter of fact Dioscorea antaly are known to induce inflammation and itching due to histamine, saponins and furanoid-norditerpene groups of compounds present in yams. In conclusion these experiments show that medaka embryos, already demonstrated to be a valuable experimental tool to analyse the toxicity due to pure cyanotoxins (Jacquet et al., 2004; Huynh-Delerme et al., 2005), crude extracts from cyanobacteria contaminating fresh water as Table 1 Sizes of sinusoidal capillaries in the liver of controls and Dioscorea antaly treated embryos.
Thickness Width
Controls (mm)
Treated (mm)
49.2 1.9 63.4 3.5
80.9 5.8 137.2 10.8
Data represent the mean – SEM of 8 replicates. Thickness as well as widths of the capillaries in treated embryos are significantly increased when compared to controls (p < 0.001).
We thank Simone Puiseux-Dao and Me´lodie Male´cot for helpful advices. This paper is a contribution to the IRD research group CYROCO UR 167. Conflict of interest The authors declare that there are not conflicts of interest.
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