Comparative teratogenicity of Chlorpyrifos and Malathion ...

Aquatic Toxicology 70 (2004) 189–200

Comparative teratogenicity of Chlorpyrifos and Malathion onXenopus laevisdevelopment

Patrizia Bonfantia, Anita Colomboa, Federica Orsia, Ilaria Nizzettoa,Manuela Andriolettib, Renato Bacchettab, Paride Manteccab,

Umberto Fascioc, Giovanni Vailatib, Claudio Vismarab,∗

a Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano Bicocca,Piazza delle Scienza 1, I-20126 Milano, Italy

b Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italyc Centro Interdipartimentale di Microscopia Avanzata (CIMA), Università degli Studi di Milano,

Via Celoria 26, I-20133 Milano, Italy

Received 4 November 2003; received in revised form 30 July 2004; accepted 15 September 2004

Abstract

The embryotoxic potential of chlorpyrifos (CPF) and malathion (MTN), two organophosphorus insecticides (OPs), wasevaluated by modified Frog Embryo Teratogenesis Assay-Xenopus(FETAX). CPF and MTN were not embryolethal even at thehighest concentration tested (6000�g/l), but both exhibited a powerful teratogenicity. The probit analysis of malformed larvap out1 coupledw rtrophiesl vel. Wes n-responsei ss©

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sub-arm-ta-oxic

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ercentages showed a TC50 of 161.54�g/l for CPF, and a TC50 of 2394.01�g/l for MTN. Therefore, CPF teratogenicity was ab5 times higher than MTN. Larvae of both exposed groups were mainly affected by ventral and/or lateral tail flexureith abnormal gut coiling. Histopathological diagnosis displayed abnormal myotomes and myocytes with marked hype

ocalized at the cell extremity, probably due to a break away of myofibril extremities at the intersomitic junction lepeculate that this muscular damage was related to inhibition of acetylcholinesterase that showed a clear concentration CPF and MTN exposed larvae. The teratogenic effects of these anti-cholinesterase compounds onXenopus laevismyogenesiuggest a possible role played by OPs on induction of congenital muscular dystrophy.2004 Elsevier B.V. All rights reserved.

eywords: Chlorpyrifos; FETAX; Malathion; Muscular damage

∗ Corresponding author. Tel.: +39 02 503 14741;ax: +39 02 503 14802.

E-mail address:[email protected] (C. Vismara).

1. Introduction

In recent decades, many pesticides have beenjected to careful investigations to assess their hful effects on the environment. While highly sble chlorinate hydrocarbons are undoubtedly ecot

166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.aquatox.2004.09.007

190 P. Bonfanti et al. / Aquatic Toxicology 70 (2004) 189–200

compounds, the hazard effects of rapidly degradedorganophosphates insecticides (OPs), widely used inagriculture and in households, are less evident. OPsability to poison insects is the result of their anti-cholinesterase activity. The usual symptoms in in-sects roughly follow the general pattern of nerve poi-soning like hyper excitability, tremors, convulsionsand paralysis that lead to death. Many other support-ing or contradicting data for the theory of OP anti-cholinesterase activity could be described, but mostevidence, at present, strongly support the view thatcholinesterase inhibition in vertebrates is analogousto what happens in insects. In both insects and ver-tebrates it is common practice to separate OP poison-ing symptoms into muscarinic, nicotinic and centralnervous. Of these, the nicotinic effect is the result ofaction on somatic nerve elements which cause an overstimulation followed by paralysis of voluntary mus-cles (Matsumura, 1975). Indeed, OP neurotoxicity me-diated by the phosphorylation and subsequent inhibi-tion of acetylcholinesterase (AChE) is most noted inmammals, humans included (Oehmichen and Besserer,1982; Finkelstein et al., 1988). Even if OPs have notbeen classified as teratogenic compounds in mice andrats (Bleyl, 1980; Robinson et al., 1986; Ruckman etal., 1999), there are growing data showing that theyare teratogenic on the grounds of experimental con-centrations in non-mammal developing embryos, suchas amphibians (Richards and Kendall, 2002) and birds(Meiniel, 1981).

m-be nts( t al.,1 o-g ion( tht thatA ent( us-c l-l ohne or-m cu-ll yeda ity(

2. Materials and methods

2.1. Chemicals and solutions for the bioassay

The organophosphorus insecticides chlorpyri-fos (O,O-diethyl O-[3,5,6-trichloro-2-pyridyl]phosphorothioate; CPF) and malathion (S-[1,2-dicarbethoxyethyl] O,O-dimethyldithiophosphate;MTN) with over 99% purity were supplied by Lab-service Analytica S.r.l, Italy. All analytical gradereagents, human chorionic gonadotropin (HCG),3-amino-benzoic acid ethyl ester (MS 222), Tri-ton X-100, 4′,6-diamidino-2-phenylindole (DAPI),phalloidin-TRITC were obtained from Sigma-AldrichS.r.l., Italy. The control FETAX solution compo-sition in mg/l was NaCl 625, NaHCO3 96, KCl30, CaCl2 15, CaSO4-2H2O 60, and MgSO4 70,pH 7.5–8.5 (Dawson and Bantle, 1987). The DeBoer-Tris (DBT) solution in mg/l was NaCl 6,900,KCl 186, CaCl2 200 buffered to pH 7.5 with 10 mMTris–HCl.

2.2. Animals

Adult males and femalesX. laevis were fromthe Centre d’elevage de Xenopes du CNRS, Rennes,France. The animals were acclimated for at least6 weeks in aquaria with dechlorinated tap water at22± 2◦C, alternating 12 h light–dark cycles and fed asemi synthetic diet from Mucedola S.r.l., Settimo Mi-l

2(

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w .A e fe-m etrid erms alet ,1 rea was

In this paper, with the aid of the modified Frog Eryo Teratogenesis Assay-Xenopus(FETAX), a pow-rful and flexible bioassay for developmental toxicaDumont et al., 1983; Bantle et al., 1990; Vismara e993; Bernardini et al., 1994), we studied the teratenic potential of chlorpyrifos (CPF) and malathMTN), comparing the rate of AChE inhibition wihe degree of the teratogenic effects. ConsideringChE is required for embryo muscular developm

Behra et al., 2002) and OPs are able to induce mular damages in mammals (Gupta et al., 1987; Karaiedde and Henry, 1993; De Bleecker et al., 1994; Jt al., 2003), we studied at histological level the malfations induced by CPF and MTN on the tail mus

ature, since previous works had shown thatXenopusaevis larvae exposed to these compounds displan abnormal tail flexure with impairment of motilVismara et al., 1996; Richards and Kendall, 2002).

anese, Italy, three times a week.

.3. Frog Embryo Teratogenesis Assay-XenopusFETAX)

We used a modified version of FETAX (Bernardint al., 1994) as follows.

.3.1. In vitro fertilisationFor a single bioassay, six females were inje

ith 900 IU while two males with 500 IU of HCGbout 16 h later, eggs obtained by massaging thales’ abdomens were put in a 140-mm plastic Pishes and then artificially inseminated with a spuspension previously obtained by mincing adult mestes in 1–2 ml of cold DBT solution (Vismara et al.993). One minute later, 30 ml of FETAX solution wedded to each Petri dish. Successful insemination

P. Bonfanti et al. / Aquatic Toxicology 70 (2004) 189–200 191

detected when the eggs were oriented with the darkanimal pole side up. A first screening performed 3 hpost fertilisation, p.f., enabled us to remove the unfer-tilised and necrotic eggs; this was followed by a sec-ond screening, 5 h p.f., stage 8, blastula, in which nor-mal cleavage was ascertained (Niewkoop and Faber,1956).

2.3.2. CPF and MTN experimental groupsTen undejellied normal blastulae from the same fe-

male were put in a 40 mm Petri dishes to make controland exposed groups. Each Petri dish contained 20 ml ofcontrol or test solution. This procedure was followedfor the six females of the bioassay. Blastulae (stage9, 8 h p.f.) were exposed to eight CPF concentrationsranging from 50 to 6000�g/l or to five MTN concentra-tions ranging from 375 to 6000�g/l. The exposure wassuspended at stage 47 of free swimming larva (120 hp.f.). The single bioassay was repeated four times forCPF and three times for MTN under the same ex-perimental conditions. During the bioassay the disheswere kept in a thermostatic chamber at 23± 0.5◦C andeach day the test solutions were renewed and the deadembryos removed. The number of dead embryos wasrecorded.

2.3.3. Data collectionAt the end of exposure time (stage 47), the num-

ber of dead embryos was calculated, while the surviv-i g/l)f al-f al-f p ofo ereg dis-c

2on-

c bryosa g tocf end-iT thet nds,w

2.4. Histological studies

Stage 47 control larvae and those exposed to CPFor MTN at 3000�g/l were fixed in 10% formalde-hyde in PBS, repeatedly rinsed in PBS, dehydratedin a graded alcohol series and embedded in paraffin.Transversal and para-sagittal 5�m sections were de-waxed, hydrated in a graded ethanol series and stainedwith Mayer’s haemalaun and eosin. Further controland exposed larvae were fixed for 2 h in modifiedKarnowsky’s liquid (2% paraformaldehyde, 0.2% glu-taraldehyde in 0.1 M PBS, pH 7.4), dehydrated ingraded alcohol series and embedded in epoxy resinEpon 812. Transversal and para-sagittal 0.5�m semi-thin sections were stained with crystal violet and basicfuchsin. Slides were examined under a light Zeiss Ax-ioplan MC 100 microscope. Images were taken witha colour digital Image Pro Plus version 4.5.1, MediaCybernetics.

2.5. Confocal microscopic analysis

Stage 47 control larvae and those exposed to CPFor MTN at 3000�g/l were anaesthetised with MS222 (100 mg/l). Whole-mount tails were fixed in 4%paraformaldehyde in PBS buffer 120 mM for 1 h andwashed in Triton X-100 (0.4% in PBS buffer 120 mM)for 20 min and then with PBS 120 mM for 15 min. Actinwas stained with 0.5�g/ml phalloidin-TRITC and nu-clei with 20�g/ml DAPI for 30 min. The tails werew sedm nfi CS-N el-b sec-t itha serw

2

CPFa 0a n,d fivep per-f ce-d -

ng larvae were anaesthetised with MS 222 (100 mor morphological examination. The number of mormed larvae as well as the frequency of single mormations was recorded. When in the control groune female the mortality and malformation rates wreater than 20%, all groups of that female werearded (Bernardini et al., 1994).

.3.4. Statistical analysisThe relationship between the CPF and MTN c

entrations and outcomes, percentage of dead emnd malformed larvae, was investigated by resortinhi-square and probit analysis (Finney, 1971). The ef-ective concentrations at 120 h p.f. are called, depng on the case, lethal (LC50) or teratogenic (TC50).he Teratogenic Index, T.I., useful in estimating

eratogenic risk associated with the tested compouas the ratio LC50/TC50 (Dawson and Bantle, 1987).

ashed in PBS 120 mM, mounted in a glycerol-baedium and stored at 4◦C in the dark. Myocyte acti

laments and nuclei were visualised with a Leica TT confocal microscope, Leica Microsystem, Heiderg, Germany. Focal series of horizontal planes of

ion were monitored for TRITC using a Ar/Kr laser wlong pass filter LP 590 and for UV using a Ar ion laith a long pass filter LP 450.

.6. AChE activity analysis

Stage 47 control larvae and those exposed tot 100, 250 and 3000�g/l and to MTN at 1500, 300nd 6000�g/l were used for AChE activity evaluatioetermined in three independent experiments fromools of five whole body larvae. Each assay was

ormed in triplicate using a modified Ellman proure (Ellman et al., 1961) with acetylthiocholine io


dide (0.075 M) as a substrate. The Ellman protocol wasadapted to quantify AChE activity inX. laevislarvae(Gindi and Knowland, 1979). Briefly, whole body lar-vae were homogenised in Tris buffer (1% Triton X-100in 0.05 M Tris–HCl, pH 7.4) at 1:10 (w/v) and cen-trifuged for 5 min at 15,000 g. The presence of thiolgroups masks the low levels of AChE causing an in-correct evaluation of enzyme activity; for this reason,before adding the substrate, a 10 min pre-incubationwith 0.33 mM 5,5′-dithio-bis (2-nitrobenzoic acid) ofextracts was necessary. Changes in optical density werenoted every 15 s for 15 min at 412 nm. Preliminary ex-periments were conducted to define conditions for sam-ple concentration required for linear rates of substrateshydrolysis. AChE activity was calculated as nmol ofacetylthiocholine iodide hydrolysed per min per gr lar-vae and the results were expressed as percentages ofAChE inhibition versus control. At the testing of thedifferences of the arithmetical means between specificgroup of samples (CPF and MTN 3000�g/l) thet-testwas applied. Conclusion about the rejection of the nullhypothesis of the equality and the differences, was doneatP< 0.001.

3. Results

Stage 35–36 embryos exposed to CPF and MTNmoved normally but immediately after they showed de-fects of neuromuscular activity such as spasms, tremorsa laters ae.

3

inT ven

Fig. 1. TC50 predicted value ofX. laevis larvae exposed to CPF,calculated by probit analysis.

at the highest 6000�g/l concentration. On the otherhand, a good concentration-response was observed inmalformed larva percentages. There was a significantdifference between control malformed larva percentageand that at 100�g/l, while 100% of the larvae were mal-formed at 750�g/l (Table 1). The percentages of mal-formed larvae investigated by probit analysis allowedto calculate a TC50 of 161.54�g/l (Fig. 1). Becausethere was no mortality even at 6000�g/l, the LC50 andthus the T.I. could not be calculated. Since T.I. wouldbe many times greater than 3, according toDawson andBantle (1987), CPF must be considered a powerful ter-atogenic compound. Unlike the control (Fig. 2A), CPFlarvae were mainly affected by ventral and/or lateral tailflexure coupled with abnormal gut coiling (Fig. 2B, C).

MTN also showed no lethality at any concentrationsused (Table 2), but like CPFs, it was able to induce mal-formations with a good concentration-response. There

TC

250 500 750 1000 3000 6000

U 149 150 90 90 180 90D 5 1 7 5 10 6M 3.4 0.7 7.8 5.5 5.6 6.7L 144 149 83 85 170 84M 90 142 83 85 170 84M 62.5** 95.3** 100** 100** 100** 100**

nd, at the highest concentrations, paralysis thateriously compromised swimming of stage 47 larv

.1. CPF and MTN embryotoxicity

The CPF embryotoxic responses are givenable 1. The compound was not embryolethal e

able 1PF embryotoxicity onX. laevis(stage 47)

Concentrations CPF (�g/l)

50 100

tilized embryos (n) 790 140 137ead embryos (n) 5 3 5ortality (%) 0.6 2.1 3.6iving larvae (n) 785 137 132alformed larvae (n) 36 10 52alformed larvae (%) 4.6 7.3 39.4*

∗ P< 0.05.∗∗ P< 0.01.


Fig. 2. Stage 47X. laevislarvae. (A) dorsal view of a control larva; (B), (C) and (D) abnormal tail flexure coupled with abnormal gut coiling;(B) dorsal view; (C) lateral view of larva exposed to CPF; and (D) lateral view of larva exposed to MTN. Bars = 3 mm.

was a significant difference between control and ex-posed groups starting from 1500�g/l, but 100% ofmalformed larvae was not reached even at 6000�g/l(Table 2). The percentages of malformed larvae inves-tigated by probit analysis allowed to calculate a TC50 of2394.01�g/l (Fig. 3) and, here too, MTN must be con-sidered a powerful teratogenic compound. Moreover,the MTNs were mainly affected by abnormal tail flex-ure, malformation easily comparable to that observedin CPFs (Fig. 2D).

3.2. Histological studies

The histological screening was done to identifythe main histopathological features of myotomes, my-ocytes and notochord. In the controls the myocyteswere normally orientated in parallel to the noto-chord, occupying the whole length of the myotomes,and were attached at regular intersomitic boundaries

Fig. 3. TC50 predicted value ofX. laevislarvae exposed to MTN,calculated by probit analysis.

Table 2MTN embryotoxicity onX. laevis(stage 47)

Concentrations MTN (�g/l)

375 750 1500 3000 6000

Utilized embryos (n) 152 93 113 113 169 117Dead embryos (n) 13 8 8 12 17 14Mortality (%) 8.5 8.6 7.1 10.6 10.1 12Living larvae (n) 139 85 105 101 152 103Malformed larvae (n) 14 11 9 23 113 98Malformed larvae (%) 10.1 12.9 8.6 22.8** 74.3** 95.1**

∗∗ P< 0.01.


Fig. 4. Paraffin sections of stage 47X. laevislarvae. (A) transversal; (B) para-sagittal sections of control larvae; (C) transversal; (D) para-sagittalsections of CPF exposed larvae (3000�g/l); (E) transversal; and (F) para-sagittal sections of MTN exposed larvae (3000�g/l). sc: Spinal cord;M: myotomes; n: notochord; m: myocytes; ib: intersomitic boundaries; nf: notochord flexure; dM: distorted myotomes; hm: horizontal myocytes;uib: undeveloped intersomitic boundaries. Bars = 100�m.

(Fig. 4A, B). In CPFs notochord flexure was evidentand myocytes had not a correct orientation, a few be-ing oriented even perpendicular to the notochord. My-otomes were smaller and seriously distorted than inthe controls, while the intersomitic boundaries wereonly partially developed (Fig. 4C, D). The patholo-gies found in MTNs were similar and easy comparableto those observed in CPFs. The notochord, myotomesand myocytes showed a phenotype entirely altered andsometimes the myocytes were oriented perpendicularlyto the notochord (Fig. 4E, F). Semi-thin sections ofcontrol larvae showed regular myocytes with normalcontractile apparatus, lipid droplets and yolk granules(Fig. 5A, B). On the contrary in CPFs and MTNs, my-ocytes were completely distorted, rich in lipid droplets,

pointed in different direction and with no clear cellularboundaries. The most outstanding pathologies were theheavy disarrangement of contractile structures and themarked hypertrophies at the cell extremities (Fig. 5C,D).

3.3. Confocal microscopic analysis

Confocal microscopic analysis of control myotomesconfirmed the normal morphology observed in paraf-fin and resin sections. The multinucleate myocytesoccupied the whole length of the myotomes and at-tached at regular intersomitic boundaries (Fig. 6A).The images showed characteristic myocytes packedwith cross-striated myofibrils (Fig. 6B). In CPFs many


Fig. 5. Semi-thin sections of stage 47X. laevismyotomes. (A) transversal; (B) para-sagittal sections of control larvae; (C) para-sagittal sectionof CPF exposed larva (3000�g/l); and (D) para-sagittal section of MTN exposed larva (3000�g/l). m: Myocytes; N: nucleus; ca: contractileapparatus; ld: lipid droplets; ib: intersomitic boundary; yg: yolk granules; aib: alterated intersomitic boundaries; mh: myocyte hypertrophies.Bars = 20�m.

dramatic alterations were present. The myocytes weresmaller, completely distorted and with contractile ap-paratus, when present, made of myofibrils that seldomshowed cross striations (Fig. 6C). This confocal analy-sis showed that the hypertrophies observed in paraffinand resin sections were due to a break away of my-ofibrils extremities (Fig. 6E). This last alteration ofthe contractile apparatus was most evident in MTNs(Fig. 6D, F).

3.4. AChE activity analysis

AChE activity inhibition was investigated inX. lae-vis larvae exposed to concentrations lower and higherthan TC50 for both CPF (TC50 = 161.54�g/l) andMTN (TC50 = 2394.01�g/l). As showed inFig. 7agood concentration-response was observed betweenCPF and MTN concentrations and percentages ofAChE inhibition. In CPFs, the inhibition was 75.71%at 100�g/l, 79.73% at 250�g/l whereas it reached94.12% at 3000�g/l. Instead, in MTNs, 58.45% ofAChE inhibition was observed at 1500�g/l, 74.26%at 3000�g/l and 95.02% at 6000�g/l. At the same

concentration of 3000�g/l, the AChE inhibition wassignificantly higher (P< 0.001) in CPF than in MTNexposed larvae.

4. Discussion

The present paper showed no embryolethal effectsof CPF and MTN inX. laevisembryos exposed fromstage 9 (8 h p.f.) to 47 (120 h p.f.), even at the highestconcentrations tested. Previous studies on this speciesfound a LC50 value of 14,600�g/l for CPF in em-bryos exposed for 96 h starting from stage 14 (16 hp.f.) (Richards and Kendall, 2002) and of 10,900�g/lfor MTN in embryos exposed for 96 h starting fromstage 2 (1.5 h cleavage initiated) (Snawder and Cham-bers, 1989). These data evidenced a relative tolerancein terms of mortality ofX. laevisembryos to OP pes-ticides. The tolerance to OPs changes from speciesto species and inside the same species, and is influ-enced by several factors such as sensitivity of the tar-get, disposition and metabolism (Chambers and Carr,1995). In X. laevistadpoles, the relative insensitivity


Fig. 6. Confocal microscopic analysis of stage 47X. laeviswhole-mount tails. (A) myotomes; (B) myocytes of control larva; (C) myotomes;(E) myocytes of CPF exposed larvae (3000�g/l); (D) myotomes; and (F) myocytes of MTN exposed larvae (3000�g/l). Red, actin stainedwith phalloidin-TRITC; blue, nuclei stained with DAPI. M: myotomes; m: myocytes; ib: intersomitic boundaries; mN: myoblast nucleus; smN:secondary myoblast nucleus; my: myofibrils; dm: distorted myocytes; mh: myocyte hypertrophies.

to OPs was ascribed to AChE resilience toward theseinhibitors (Shapira et al., 1998), while in Bufo are-narum the tolerance to OPs was related to a reduc-tion in glutathione content and increase in glutathione

S-transferase activity (Anguiano et al., 2001). More-over, OP acute toxicity depends on the activation tooxon metabolites mediated by cytochrome P450 andX. laevisdevelopmental stages considered in this study


Fig. 7. Inhibition of AChE activity in stage 47X. laevislarvae after exposure to CPF (�) and MTN (�). Values were based on the assumptionthat control larvae had 0% of inhibition of AChE activity. Each value represents the mean± S.D. obtained from three independent assays carriedout in triplicate on five pools of larvae. When not given, S.D. bars were smaller than the used scale. Key: ***P< 0.001 between the markedpoints.

lack a competent metabolic activation system (Fort etal., 1988; Bantle et al., 1999), which starts to be suffi-ciently expressed only after 120 h p.f. (Colombo et al.,1996).

Beside a high tolerance in terms of mortality, wefound that both compounds caused 100% of malformedlarvae at relatively low concentrations among thosetested.X. laevisembryos showed different ranges ofsensitivity towards the pesticides examined and CPFresulted nearly 15 times more teratogenic than MTN.In addition to these results, a substantial differencein AChE inhibition between the two compounds wasfound in this study. The degree of AChE inhibitionin CPFs and MTNs showed a good concentration-response and at 3000�g/l, a statistically significant dif-ference was found between CPFs and MTNs, with analmost complete inhibition in CPFs. These differencesin CPF and MTN toxicity could be explained by sev-eral contributing factors.Lund et al. (2000)reporteda higher half-life and octanol/water partition coeffi-cient for CPF than MTN and, according toSerranoet al. (1997), this can influence a different persis-tence of the two compounds in the test solutions, aswell as their levels of accumulation in tissues.Booneand Chambers (1997)reported that AChE target en-zyme sensitivity is the main factor of high CPF toxic-ity. Moreover, the sterical hindrance, smaller in CPFthan in MTN, would make CPF more accessible tothe AChE active centre justifying its higher inhibitorypotency.

A strong correlation between AChE inhibition andmalformation percentages was observed both for CPF(r2 = 0.97,P< 0.01) and MTN (r2 = 0.90,P< 0.01) and,although the CPF and MTN embryotoxicity inX. laeviswas at different concentrations, similar terata occurred.

Larvae scored as malformed, showed often grossalterations in the tail flexure coupled with a great de-crease of neuromuscular activity, and these results arewell comparable to those of other authors (Snawderand Chambers, 1989; Vismara et al., 1996; Richardsand Kendall, 2002). It is well known that in non-targetorganisms, such as amphibians, fishes, birds and mam-mals, AChE inhibition is the primary manifestation oftoxicity during OP exposure (Thomson et al., 1991;Sultatos, 1994; Calumpang et al., 1997; Fulton and Key,2001) and that OP compounds produce typical signs ofanti-cholinesterase toxicity such as complex posturingmovements, body shaking or skeletal muscle fascicula-tion associated with muscle fibre damages in mammals(Gupta et al., 1987; Karalliedde and Henry, 1993; DeBleecker et al., 1994; John et al., 2003). The abnor-mal tail flexure observed in CPFs and MTNs could bethe consequence of the cholinergic phase, where theAChE inhibition causes repetitive firing of muscle fi-bres leading to the axis tail folding and, at high con-centrations, to paralysis. Previous data on lower ver-tebrates showed that newly hatched catfish larvae ex-posed to MTN developed deformed tail, as a conse-quence of uncontrolled and continuous contraction ofthe tail musculature (Lien et al., 1997). This hypothe-


sis was confirmed byBehra et al. (2002)who noticedthat the first movements of zebrafish embryos homozy-gous for AChE gene mutation were characterised bytail twitching. During the further development, mutantsshowed severe myopathy characterised by disruption ofmyocyte myofibrils due to the lack of AChE activity. Onavian embryos OPs induced gross malformations, suchas variable degrees of vertebral fusion, probably due tothe muscular cell sarcolemma depolarisation, followedby uninterrupted muscle contractions. Therefore, theconcurrent vertebral organogenesis was seriously dis-torted (Meiniel, 1981). Also in X. laevis, Snawder andChambers (1993)found abnormal notochord morpho-genesis after MTN exposure.

Tail flexures that we observed in exposed embryoswere histologically characterised by abnormal noto-chord curvature and disorganised myotomes. Sinceearly in the normal amphibian myogenesis, myotomalcells change in position from perpendicular to par-allel relative to axial notochord (Kielbowna, 1981;Daczewska, 2001), a possible mechanism for histopa-tological myotome damages in CPFs and MTNs maybe related to an early alteration of the normal my-otomal cell orientation. Therefore, myocytes were per-pendicular rather than parallel to the axial notochord(Fig. 4C, E). Moreover, like in zebrafish mutants lack-ing of AChE activity (Behra et al., 2002), the extensiveand marked hypertrophies localised at myocyte extrem-ity level, as well as myofibril disorders (Fig. 5C, D),could be due to a myofibril break away and a pertur-bI om-e thet iticj aint on-t

tusc ost-s ad-i edm ry,1 -l celldT clen er( ch

specifically remove Z-disks in mammalian skeletalmuscles. A similar mechanism was also observed invenom-induced muscle degeneration (Harris et al.,2003). The snake venom determined a depolarisationof the muscle fibres followed by a hyperconcentrationof Ca2+ into the cytosol. Since the rapid elevation ofCa2+ was responsible for the activation of intermediatefilament specific proteases (Nelson and Traub, 1983), ahydrolysation of desmin and titin was consequent, andthis represents an early feature of stress- and disease-related muscle degeneration (Harris et al., 2003). Inparticular desmin, which is the major intermediate fil-ament in skeletal muscle, is crucial to interlink adjacentsarcomeres, forming a collar around the Z-disks, andto strengthen the tethering of myofibrils to the plasmamembrane. The loss of desmin would lead to the lossof register between adjacent sarcomeres and would ex-pose titin to hydrolytic attack leading to the disaggre-gation of the sarcomeres (Harris et al., 2003). In X. lae-vis myotomal muscle with truncated forms of desmin,Cary and Klymkowsky (1995)reported structural de-fects at the ISJ, confirming the important role of inter-mediate filaments in the organisation and stabilisationof myofibril-membrane attachment sites. The abnormaldevelopment of the ISJ would also determine an in-correct differentiation of the dystrophin–glycoproteincomplex exhibiting a phenotype very similar to thatobserved in myocytes of mdx mouse, which lacks ofdystrophin (Cary and Klymkowsky, 1995).

In conclusion the CPF and MTN teratogenic effectso os-s s oni net-i

A

AR2 .

R

A .M.,arlyysiol.

ation of myocyte contractile apparatus (Fig. 6E, F).n detail, our hypothesis is that the terminal sarcric actin filaments may lose their connection with

rans–membrane protein complex at the intersomunction (ISJ), a highly specialised sarcolemma domhat ensure the integrity of the muscle fibres during craction (Peng and Chen, 1992).

The perturbation of myocyte contractile apparaould be ascribed to an overstimulation of the pynaptic membrane, caused by AChE inhibition, leng to excessive Ca2+ influx as reported in OP treat

ammals (Wecker et al., 1986; Karalliedde and Hen993; De Bleecker et al., 1994). Alteration of intracel

ular Ca2+ homeostasis has been associated witheath in a variety of system (Orrenius et al., 1989).he involvement of calcium as the mediator of musecrosis has been described byLeonard and Salpet1979) who referred Ca2+-activated proteases whi

bserved duringX. laevismyogenesis suggest a pible role played by anti-cholinesterase compound

nduction of congenital muscular dystrophy not gecally inherited.

cknowledgements

This research was supported by grant F001–2002 to A.C. and by grant FIRB 2001 to C.V

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