ilable at ScienceDirect

Toxicon 55 (2010) 1255–1262

Contents lists ava

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

Histopathological effects of [D-Leu1]Microcystin-LR variants on liver,skeletal muscle and intestinal tract of Hypophthalmichthys molitrix(Valenciennes, 1844)

Maria Fernanda Nince Ferreira a,*, Veronica Moraes Oliveira b, Rhaul Oliveira a,Priscila Vieira da Cunha a, Cesar Koppe Grisolia a, Osmindo Rodrigues Pires Junior b

a Departamento de Genetica e Morfologia, Instituto de Ciencias Biologicas, Campus Darcy Ribeiro, Universidade de Brasılia, Asa Norte, Brasılia, DF, Brazilb Departamento de Fisiologia, Instituto de Ciencias Biologicas, Campus Darcy Ribeiro, Universidade de Brasılia, Asa Norte, Brasılia, DF, Brazil

a r t i c l e i n f o

Article history:Received 21 May 2009Received in revised form26 January 2010Accepted 29 January 2010Available online 6 February 2010

Keywords:[D-Leu1]Microcystin-LRHypophthalmichthys molitrixHistopathology

* Corresponding author. Universidade de BrasıliaRibeiro - Asa Norte Instituto de Biologia LaboratoGenetica, Bloco 8, Brazil. Tel.: þ55 61 3307-2169; fax

E-mail address: mfnf@unb.br (M.F. Nince Ferreir

0041-0101/$ – see front matter � 2010 Elsevier Ltddoi:10.1016/j.toxicon.2010.01.016

a b s t r a c t

This study evaluated the effects of [D-Leu1]Microcystin-LR variants, by the exposure ofHypophthalmichthys molitrix to Microcystis aeruginosa NPLJ4. Fish was placed in aquariumsand exposed to 105 cells mL�1. For 15 days, 05 individuals were removed every 05 days,and tissue samples of liver, skeletal muscle and intestinal tract were collected for histo-pathologic analyses. Following exposure, those surviving were placed in clean water for 15days to evaluate their recovery. A control without toxins was maintained in the sameconditions and exhibited normal histology and no tissue damage. In exposed fish, sampleswere characterized by serious damages that similarly affected the different organs, such asdissociation of cells, necrosis and haemorrhage. Samples showed signs of recovery butsevere damages were still observed. The results should be valuable to analyze the potencyof microcystin toxicity and to help in the diagnosis of fish deaths.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Intense anthropogenic activity in river basins under-mines the quality of water resources, particularly withregard to eutrophication. Fish deaths have been reported inconjunction with toxic cyanobacterial blooms in naturalsystems and in farms. In these rearing systems, theconsequent nutrient enrichment of the aquatic environ-ment may lead to frequent cyanobacterial blooms.

Cyanobacteria can produce toxins and the mostcommon of them is the hepatotoxic cyclic peptide micro-cystin (MC), which possesses among 60 described variants(Sivonen and Jones, 1999). Microcystins act as powerfulinhibitors of the serine and threonine residues of the

, UnB Campus Darcy´ rio de Morfologia e: þ55 61 3273-3942.

a).

. All rights reserved.

phosphatase proteins PP1, PP2A and PP3A and PP6(Mackintosh and Mackintosh, 1994; Runnegar et al., 1995;Toivola and Eriksson, 1999). Microcystins have beenshown to be highly organotropic for the liver and gastro-intestinal tract, as indicated by strong histopathologicallesions observed in laboratory data in fishes and othergroups (Claeyssens et al., 1995; Kotak et al., 1996; Fischerand Dietrich, 2000; Botha et al., 2004; Mohamed andHussein, 2006). The dissociation of the hepatic cytoskel-eton and, subsequently, progressive liver necrosis andapoptosis has been widely reported in fish, as well ashepatic tumours and strong hepatic haemorrhages(Tencalla et al., 1994; Fischer et al., 2000).

One important aspect is the particular susceptibility ofspecies such as Hypophthalmichthys molitrix (Valenciennes,1844), a cyprinid known as silver carp, with commercialand aquacultural importance. This phytoplanktivorousspecies was introduced around the word for control of algalblooms in impoundments for its ability to clean clogging

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–12621256

algae from waters. Therefore, this fish may be exposed tomicrocystins through their own feeding activities, ingestinga large amount of cyanobacteria (Xie et al., 2004; Chenet al., 2006).

The purpose of this study is to evaluate the chroniceffects of Microcystin toxins on the structure of thealimentary system, liver and skeletal muscle, by the chronicexposure of H. molitrix to Microcystis aeruginosa NPLJ4 cellsin water, in a system similar to that observed in a naturalhabitat.

2. Materials and methods

2.1. Culture of cyanobacteria and source of experimentalanimals

Unialgal cultures of M. aeruginosa NPLJ4 were carriedout in the Laboratory of Toxinology, University of Brasilia.Examples of silver carp (H. molitrix) measuring 12 cm werecollected in Lake Paranoa, Brasilia-DF. Animals weredistributed in thermostatically controlled aquariums of90 L (30 cm � 60 cm � 50 cm) with filtrated and dechlor-ined tap water, acclimatized for 20 days at a temperature of25 � 2 �C, photoperiod of 12 h and continuous oxygenationwith submerged pumps. Fish were feed with fish chow.

2.2. M. aeruginosa NPLJ4 toxins characterization – HPLC andmass analyses

One gram of freeze-dried culture cells were extracted (3times) in 100% methanol (20 mL/g). The methanolicextracts were evaporated to dryness at 40 �C, and theresidues were resuspended in deionized water. The samplewas purified with a C18 cartridge and analyzed in a Shi-madzu LC-10A HPLC equipped with a photodiode arraydetector SPD MXA-10. The chromatography was carried outunder at isocratic conditions using a reverse phase C18column (Synergi 4m Fusion-RP 80 (250 � 4.60 mm; Phe-nomenex)), mobile phase of 20 Mm ammonium formiate,pH 5,0 and acetonitrile (7:3, v:v) per 35 min. The identifi-cation of the microcystins produced by M. aeruginosa NPLJ4was performed by comparison of the spectrogram simi-larity index of standard microcystin-LR (SIGMA, CO) in theabsorbance range of 200–300 nm.

The microcystin molecular mass were determinate byUltraflex II� TOF/TOF (Bruker, Bremen, Germany). Aliquotsof lyophilized toxins were dissolved in Milli-Q water (TFA0.1%) and mixed with a saturated matrix solution ofa-cyano-4-hydroxycinnamic acid (1:3, v/v) and directlyapplied onto a target (AnchorChip�, Bruker Daltonics). Themass spectrometry was operating in reflector mode forMALDI-TOF or LIFT mode for MALDI-TOF/TOF fully auto-mated using the FlexControl� software. Calibration of theinstrument was performed externally with [MþH]þ ions ofangiotensin I, angiotensin II, substance P, bombesin, insulinb-chain and adrenocorticotropic hormones (clip 1–17 andclip 18–39). Each spectrum was produced by accumulatingdata from 200 consecutive laser shots. Those sampleswhich were analyzed by MALDI-TOF were additionallyanalyzed using LIFT TOF/TOF MS/MS from the same target.

2.3. Exposure of silver carp (H. molitrix) to cells ofM. aeruginosa

At the end of the acclimatization period, animals weresubmitted to a volume of 840 mL culture medium containingcolonies of M. aeruginosa NPLJ4 at the end of the exponentialgrowing phase. The concentration of cells in the aquariumwas the equivalent of 105 cells per mL. During the initialperiod of 15 days, 5 (five) individuals were removed at 5-dayintervals and sacrificed by anaesthesia using injectable lido-cain 5% applied in the central nervous system. Aquaria werekept continuously aerated, temperature was maintained at25�1 �C, pH w7.0 with a photoperiod of 12 h light and 12 hdarkness. Every day the water was changed and the sameculture volume of M. aeruginosa was added, aiming tomaintain cell concentration in contact with animals. Fiveindividuals were assigned to the histological analyses in lightmicroscopy. Samples of intestinal tract (I), liver (L) and stri-ated muscle (M) were observed. The remaining animals wereremoved to another free cell aquarium and, after fifteen andseventeen days, 5 (five) individuals were collected andsacrificed following the same previously mentioned meth-odology to evaluate tissue regeneration by light microscopy.Animals that were not exposed to cell culture formed thecontrol group, and were collected under the same conditions.Histological material for the evaluation of the effect of cya-notoxins on the morphology of tissues and organs was fixedby means of tamponed formaldehyde 10%, processedaccording to the routine for light microscopyand stained withHaematoxylin and Eosin (H and E). The system forcapturing images consisted of camera CCD-Iris and captureplate PixelView-Station, Image Pro Express-4.0, MEDIACY-BERNETICS, LP and Scopephoto.

3. Results

3.1. Microcystis toxins characterization

M. aeruginosa NPLJ4 culture extract of showed thepresence of 5 microcystins that were identified by lettersA–E (Fig. 1), the chromatographic fraction C is the majortoxin within 89.91% of all microcystins detected.

The mass spectrometry analyses on reflective modeshowed the mass components at 1071.64 Da, 1023.62 Da,1037.69 Da, 1071.64 Da and 1023.64 Da for chromato-graphic fractions A, B, C, D, and E respectively. The massspectrometry analyses in lift mode confirms the presenceof microcystins by showing in the fragment pattern the ion135.03, a ADDA fragment exclusive from microcystin toxins(Fig. 2). The chromatographic fraction C was identity as [D-Leu1]MC-LR (Fig. 2), by also showing the fragment 595.26(M þ Hþ) corresponding to Mdha–Leu–Leu–MeAsp–Arg(Matthiensen et al., 2000), the others 4 microcystins hadalso shown the same fragment suggesting that all are [D-Leu1]MC-LR variants (Data not shown). For the masscomponents at 1023 Da and 1071 Da we suggest as des-methyl [D-Leu1]MC-LR and Dimethyl[D-Leu1]MC-LR,respectively. The concentration of [D-Leu1]MC-LR itself forthe assays was estimated in 11 mg for 840 mL culturemedium containing colonies of M. aeruginosa NPLJ4 at theend of the exponential growing phase.

Fig. 1. Chromatogram profiles of M. aeruginosa NPLJ4 culture extract showing 5 microcystins identified of A–E (on left), Spectrogram profile (range 200–300 nm)of respectively chromatogram fraction (on right).

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–1262 1257

3.2. Histology

3.2.1. ControlControl fish presented normal histology in all tissues and

organs examined, without any pathological change observed(Fig. 3). Control H. molitrixexhibited normal liver organized ina tubule sinusoidal pattern and consisting of parenchymacells, mainly hepatic cells, arranged as tubules with inter-lacing sinusoids opening in central veins, without hexagonalsubdivisions of parenchyma (liver lobes). The hepatic cells’nuclei had a single peripheral nucleolus. Hepatic tubules andexcretory passages were closely related. Bile ducts were welldisplayed, with cubic to cylindrical coating epithelium andevident brush border. Connective tissue and smooth musclewere still presented in the wall. (Fig. 3A)

The intestine hollow body was formed by four concen-tric layers: mucosa, submucosa, muscle and serosa. Themucosa was covered by a simple cylindrical epitheliumwith brush border and goblet cells. Under the epithelium,loose connective tissue with blood vessels and smoothmuscle fibres were evident. (Fig. 3B) A thin submucosaconsisted of moderately dense connective tissue. Themuscle layer was formed by smooth muscle. Outside, theorgan was coated by connective tissue and mesothelium,called the serosa layer.

Striated skeletal muscle was composed of cylindricalfibres, broad and elongated and well bounded by

sarcolemma. Several nuclei in peripheral position and crossstriations were evident. (Fig. 3C)

The toxic effects of [D-Leu1]MC-LR itself and variantsobserved on liver, muscle and intestinal tract of H. molitrixwere severe, while no fish mortality was observed in thecontrol and dose-administered fish during the exposureperiod and subsequent detoxification. In contrast tocontrol, histopathological effects were observed for all fishin the experimental group as described below.

3.2.2. 05 days of exposureAfter 05 days of exposure, contaminant-induced

changes in the structural organization of liver wereobserved, being initially restricted to the peripheral region.An apparent lysis of hepatic cells’ membranes (necroticcells) was observed associated with clumping of the chro-matin, pale staining of the cytoplasm, and deteriorizationof the cell. Localized haemorrhages were observed alongsinusoids, which were dilated like other vessels. Erythro-cytes presented irregular forms and micronuclei. Bile ductdegenerative changes, such as exfoliation of cells into thetubular lumen, anomalous nuclei and brush border notevident were observed involving epithelial cells. Macro-phages were abundant.

In the intestinal tract it was observed that in muscularlayers cells were presented separated from each other dueto the loss of intracellular adhesion. In connective tissue,

Fig. 2. Spectrogram profile in lift mode showing the fragment pattern of the microcystin chromatogram fraction C, suggesting the identity as [D-Leu1]MC-LR.

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–12621258

small blood vessels had extravasation of blood cells causingpoints of haemorrhage. A large quantity of macrophageswas found throughout the tract, with more in the looseconnective tissue below the epithelium. The columnarepithelium cells showed loss of adhesion between them.The brush border, characteristic of the absorptive epithe-lium, was not evidenced by staining with eosin.

In skeletal muscle, cells increased the intercellularspaces and the cytoplasm was scarcely stained with eosin,with no evident cross striation. Haemorrhages wereobserved in the connective tissue between the fibres.

3.2.3. 10 days of exposureTissue abnormalities detected were more advanced in

fish sacrificed after 10 days of exposure, including necrosisand a contact loss between cells, which was moreconspicuous and involved larger areas (Fig. 3D). Hepaticcells showed vacuolization in the cytoplasm and frag-mented nuclei. Sinusoid lumen was much larger than thatof control fish, changing the form of the central vein andshowing extensive haemorrhaging was observed too in

Fig. 3. Histopathological effects [D-Leu1]MC-LR itself and variants observed in livepresented normal histology in (A) liver, (B) intestinal tract and skeletal musclehaemorrhaging in liver (D), intestinal epithelium necrosis (F) and contact loss of skcongestion of blood vessels in liver (E) and increased number of granulocytes in ict ¼ connective tissue; ept ¼ epithelium; h ¼ haemorrhaging; mc ¼ muscle cell; n

arterioles and veins, with loss of epithelium cells. Gran-ulocytes and macrophage were more evident in connectivetissue involving vessels and bile duct.

The erythrocyte cells varied greatly in size and shapeand were found in large quantity. The bile duct showedsmooth muscle cells spaced because of loss of adhesion.The epithelium missed the characteristic brush border,presented spacings and few attached cells. Proliferation ofcells in the bile duct was evident. Lesions were notrestricted to the peripheral region.

In the intestinal tract serious deformities in bloodvessels and smooth muscle layers were observed. Theepithelium showed separated cells, with no clear bound-aries, some in necrosis. Already in the submucosa hae-morrhaging vessels were noted, as well as spacing betweenthe cells and large amounts of macrophage. (Fig. 3F)Thesmooth muscle fibres lost their normal architecture,showing quite clear nuclei and fragmented cells. Thesemuscle cells had poor boundaries and amorphous cyto-plasm. Between muscle layers signs of haemorrhage werenoted. The serosa layer was preserved.

r, intestinal tract and muscle of H. molitrix. H and E-stained sections control(C). After 10 days of exposure fishes showed large sinusoid lumen and

eletal muscular cells (H). After exposure signs of recovery were observed asntestine (G) but skeletal muscle showed no recovery (I). cv ¼ central vein;

and arrows ¼ sings of necrose; s ¼ sinusoids.

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–1262 1259

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–12621260

In skeletal muscle, cells were spaced, indicating contactloss. Myofibrils, the smallest units of the contractile mate-rial, were not visible with the light microscope. Haemor-rhaging vessels were found. Signs of necrosis and nuclearfragmentation were found throughout the tissue analysis.(Fig. 3H).

3.2.4. 15 days of exposureBy 15 days, tissue damage was more advanced. In

general, stains were poor. Liver showed haemorrhagingand some sinusoids were completely destroyed. Theparenchyma presented necrosis and vacuolization on bileduct epithelial cells. The vessels had even greater changesand an increase in haemorrhaging was noted. The paren-chyma has become a set of poorly organized cells. Thesinusoids no longer present a structure provided by theboards of hepatic cells which outlined its way. Bile duct andsmooth muscle cells showed more evident lesions.

In the intestinal tract, muscle layers presented loss oftheir architecture and necrosis of fibres was highlighted.The simple cylindrical epithelium with goblet cells andbrush border showed the same pattern of injury found at 10days. Submucosa and serous layers presented haemor-rhaging vessels, spacing between cells and large amountsof macrophage.

In slides examined, the individuals exposed for 15 daysshowed the first signs of fragmentation of muscle fibres.The changes observed include swelling, pale staining of thecytoplasm, lysis with release of cells contents.

3.2.5. RecoveryAt the end of the experiment, after a period without

exposure to cyanotoxins, all organs presented signs ofrecovery, especially shown by general aspects such ascongestion of blood vessels, a better stain with H and E andincreased number of granulocytes. Hepatic cells, skeletalfibres and epithelium cells had well defined nuclei andcytoplasm. In spite of indications of extensive repair, severetypes of damage like haemorrhage and necrosis were stillobserved. Especially in liver, most ducts also showeddamage similar to that found during exposure. The sinu-soids recovered their structure in part, but still presenteda large lumen in the periphery of liver. In skeletal muscleand intestinal tract lesions in blood vessels showed norecovery (Fig. 3E, G, I)

4. Discussion

Matthiensen et al. (2000) estimated that the minimumlethal dose by mouse bioassay was similar for [D-Leu1]MC-LR and MC-LR, 100 mg kg per body wt. This data indicatesthat, as microcystin-LR, [D-Leu1]MC-LR is among themicrocystins of highest toxicity (Rinehart et al., 1994;Sivonen and Jones, 1999). The occurrence of microcystins-LR desmethyl and dimethyl variants as also been reportedby Bateman et al. (1995) and Izaguirre et al. (2007),although there are no toxicity studies with these kind ofvariants.

Control H. molitrix exhibited normal histology organizedas described in the literature (Brusle and Gonzalez, 1996;Hampton et al., 1988).

After oral administration of hepatotoxin, histopathologywas characterized by a time-dependent rounding as observedduring the exposure period and according to other researchinvestigating toxicology of microcystin-LR in fish (Kotak et al.,1996; Fischer and Dietrich, 2000).

As observed by Palikova et al. (2004) and Fischer andDietrich (2000), for another cyprinid Cyprinus carpio L.,histopathologic changes were presented in the liver andgastrointestinal tract and increased in severity with timepost dosing. In contrast to our results, no overt grosshistopathological changes were observed in muscle. It waspossible that the different results were dose related and/orshowed an effect of total time of exposure, since time oftoxicant exposure, concentration and frequency wereconsidered potentially important factors (Fleeger et al.,2003). In liver, detoxification occurred by the uptake bymulti-specific bilic acid transporters and conjugation toglutathione, then being transported to kidneys and intes-tine for excretion (Ito et al., 2002; Gehringer et al., 2004;Mohamed and Hussein, 2006). Toxins presumablyreached the intestine via the bile system, according to ourresults, which demonstrated increased damage with timein intestinal tract. In Oncorhynchus mykiss and tilapia theability of microcystin to inhibit phosphatase protein wasverified, indicating the molecules were biochemicallyactive in microcystin excreted in the bile and faeces of fishpoisoned (Bury et al., 1998; Kotak et al., 1996; Best et al.,2003; Soares et al., 2004; Mohamed and Hussein, 2006).Histopathology portrays the rapid accumulation of micro-cystin in the tissue of the liver until its disposal through therecipients of bile acids and was related to the bile ductlesions. The proliferation of cells in the bile duct was alsofound in this study, as observed by Fournie and Courtney(2002).

Of interest was the fact that the histopathologic effectsobserved reflected the hypothesis that [D-Leu1]microcys-tin-LR variants may affect the general process of proteinsynthesis in the cells of different tissues, not only in hepaticcells, since there was visible damage on striated musclecells, connective submucosa and loss of adhesion betweencells in the intestine. Once inside the cells, in the case ofliver hepatic cells, it promoted disorganization of interme-diate filaments and actin known as polymers protein,components of cellular cytoskeleton (Runnegar andFalconer, 1986; Fischer et al., 2000). As observed inexposed individuals, the sinusoids have several haemor-rhages, evidencing the loss of contact with hepatic cells and,in consequence, increasingly dilated lumen as exposureprogresses. Central veins and other blood vessels wereincreasingly deformed, with disruption of the endothelium.The effects found in the intestinal mucosa and its musclelayers were similar to those found in studies with othergroups (Ito et al., 2002; Botha et al., 2004). Haider et al.(2003) and Dawson (1998) demonstrated the destabiliza-tion of the cytoskeleton of muscle fibres. The erythrocytesseem to change in size and shape and were found in varyingquantities, as reported by Kopp and Hetesa (2000). In ourstudies it was also possible to detect micronuclei in eryth-rocytes, which was an important indication of the geno-toxicity of the toxin, and has been previously described inmice (Rao and Bhattacharya, 1996; Rao et al., 1998).

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–1262 1261

The recovery period of 17 days suggested insufficienttime for regeneration of organs affected by the toxin.Fournie and Courtney (2002) indicated that in advancedstages of regeneration, cells presented rapid multiplication.Evidence of attempts at recovery was also found in thepresent study, such as granulocytes, which increased innumber.

Kotak et al. (1996) also considered that microcystin-LRdid not cause massive haemorrhage in fish liver. Wedemonstrated by histopathological analyses that haemor-rhage was a prominent feature in H. molitrix. We also believethat the ultimate cause of death in fish may be due to severepathologies such as necrosis, and to haemorrhagic shockresulting from loss of the architecture of different organs,according to other authors (Rabergh et al., 1991; Kotak et al.,1996; Jacquet et al., 2004). Both these severe types ofdamage were easily observed in H. molitrix in the short termand could determine the death of the individuals, since thedegenerative processes resulting from exposure might beonly partially reversed by the withdrawal of the toxin, asevidenced by regenerative processes. Similar effects wereobserved by others in different studies (Xie et al., 2004;Chen et al., 2006; Li et al., 2007; Qiu et al., 2007).

From these data comparisons with other fish speciescould be made in an attempt to establish a pattern ofinjuries to be used in diagnosis. In conclusion, the resultspresented in this paper should be valuable to analyze thepotency of microcystins toxicity and to help in diagnosis offish deaths. The histopathological analyses allowed a betterdiagnosis to be made and made a more accurate assess-ment of damage to the state of health of individuals,including the effects on other tissues, such as muscles andintestine.

Acknowledgements

The authors are grateful to Fernando Luıs do R. M.Starling for providing the fishes for this study; to theLaboratory for Analysis of Water, Department of Civil andEnvironmental Engineering, University of Brasilia for gentlygive a sample of Microcystis aeruginosa NPLJ4 for cultive; toEMBRAPA- CENARGEN Mass Spectrometry Laboratory forthe mass spectrometry analysis and to FINATEC for financialsupport.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

References

Bateman, K.P., Thibault, P., Douglas, D.J., White, R.L., 1995. Mass spectralanalyses of microcystins from toxic cyanobacteria using on-line chro-matographic and electrophoretic separations. Journal of Chromato-graphy A 712, 253–268.

Best, J.H., Eddy, F.B., Codd, G.A., 2003. Effects of Microcystis cells, cell extractsand lipopolysaccharide on drinking and liver function in rainbow troutOncorhynchus mykiss Walbaum. Aquat. Toxicol. 64, 419–426.

Botha, N., Ven de Venter, M., Dowing, T.G., Shephard, E.G., Gehringer, M.M.,2004. The effect of intraperitoneally administered microcystin-LR onthe gastrointetinal tract of Balb/c mice. Toxicon 43, 251–254.

Brusle, J., Gonzalez, I.A.G., 1996. The structure and function of fish liver. In:Munshi, J.S.D., Dutta, H.M. (Eds.), Fish Morphology. Science PublihersInc., New York, pp. 77–93.

Bury, N.R., Newlands, A.D., Eddy, F.B., Cood, G.A., 1998. In vivo and in vitrointestinal transport of H-microcystin-LR, a cyanobacterial toxin, inrainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 42, 139–148.

Chen, J., Xie, P., Zhang, D., Ke, Z., Yang, H., 2006. In situ studies on thebioaccumulation of microcystins in the phytoplanktivorous silvercarp (Hypophthalmichthys molitrix) stocked in Lake Taihu with densetoxic Microcystis blooms. Aquaculture 261, 1026–1038.

Claeyssens, S., Francois, A., Chedeville, A., Lavainne, A., 1995. Microcystin-LR induced an inhibition of protein synthesis in isolated rat hepato-cytes. Biochem. J 306, 693–696.

Dawson, R.M., 1998. The toxiology of microcystins. Toxicon 36, 953–962.Fischer, W.J., Dietrich, D.R., 2000. Pathological and biochemical charac-

terization of microcystin-induced hepatopancreas and kidneydamage in Carp (Cyprinus carpio). Toxico. Appl. Pharm. 164, 73–81.

Fischer, W.J., Hitzfeld, B.C., Tencalla, F., Eriksson, J.E., Mikhailov, A.,Dietrich, D.R., 2000. Microcystin-LR toxicodynamics, inducepathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (Oncorhynchus mykiss). Toxicol.Sci. 54, 365–373.

Fleeger, J.W., Carman, K.R., Nisbet, R.M., 2003. Indirect effects ofcontaminants ecosystems. Sci. Total Environ. 317, 207–233.

Fournie, J.W., Courtney, L.A., 2002. Histopathological evidence of regen-eration following hepatotoxic effects of the cyanotoxin micocystin-LRin the hardhead catfish and gulf killifish. J. Aquat. Anim. Health 14,273–280.

Gehringer, M.M., Shephard, E.G., Dowinig, T.G., Wiegand, C., Neilan, B.A.,2004. An investigation into the detoxification of microcystin-LR bythe glutathione pathway in Bald/c mice. Int. J. Biochem. Cell B. 36,931–941.

Haider, S., Naithani, V., Viswanathan, P.N., Kakkar, P., 2003. Cyanobacterialtoxins: a growing environmental concern. Chemosphere 52, 1–21.

Hampton, J.A., Lantz, R.C., Goldblatt, P.J., Lauren, D.J., Hinton, D.E., 1988.Functional units in rainbow trout (Salmo gairdneri, Richardson) liver:II. The biliary systems. Anat. Rec. 221, 619–634.

Ito, E., Takai, A., Kondo, F., Masui, H., Imanishi, S., Harada, K.I., 2002.Comparison of protein phosphatase inhibitory actiity and apparentetoxicity of microcystins and relates compounds. Toxicon 40,1017–1025.

Izaguirre, G., Jungblut, A.-D., Neilan, B.A., 2007. Benthic cyanobacteria(Oscillatoriaceae) that produce microcystin-LR, isolated from fourreservoirs in southern California. Water Res. 41, 492–498.

Jacquet, C., Thermes, V., De Luze, A., Puiseux-Dao, S., Bernard, C., Joly, J.,Bourrat, F., Edery, M., 2004. Effects of microcystin-LR on the devel-opment of medaka fish embryos (Oryzias latipes. Toxicon 43, 141–147.

Kopp, R., Hetesa, J., 2000. Changes of haematological indices of juvenilecarp (Cyprinus Carpio L.) under the influence of natural populations ofcyanobacterial water blooms. Acta.Vet. Brno. 69, 131–137.

Kotak, B.G., Semalulu, A., Fritz, D.L., Prepas, E.E., Hrudey, A.E., Coppock, R.W.,1996. Hepatic and renal pathology of intraperitoneally administeredmicrocystin-LR in rainbow trout (Oncorhynchus mykiss). Toxicon 34,517–525.

Li, L., Xie, P., Li, S., Qiu, T., Guo, L., 2007. Sequential ultrastructural andbiochemical changes induced in vivo by the hepatotoxic microcystinsin liver of the phytoplanktivorous silver carp Hypophthalmichthysmolitrix. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 146, 357–367.

Mackintosh, C., Mackintosh, R.W., 1994. The inhibition of protein phos-phatases by toxins: implications for health and an extremely sensitiverapid bioassay for toxin detection. In: Codd, G.A., Jefferies, T.M.,Keevil, C.W., Potter, E. (Eds.), Detection Methods for CyanobacterialToxins. The Royal Society of Chemistry, Cambridge, UK, pp. 90–99.

Matthiensen, A., Beattie, K.A., Yunes, J.S., Kaya, K., Codd, G.A., 2000. [D-leu]Microcystin-LR, from the cyanobacterium Microcystis RST 9501and from a Microcystis bloom in the Patos Lagoon estuary, Brazil.Phytochemistry 55, 383–387.

Mohamed, Z.A., Hussein, A.A., 2006. Depuration of microcystins in tilapiafish exposed to natural populations of toxic cyanobacteria: a labora-tory study. Ecotox. Environ. Safe. 63, 424–429.

Palikova, M., Navratil, S., Tichy, F., Sterba, F., Marsalek, B., Blaha, L., 2004.Histopathology of carp (Cyprinus carpio L.) larvae exposed to cyano-bacteria extract. Acta Vet. Brno. 73, 253–257.

Qiu, T., Xie, P., Ke, Z., Li, L., Guo, L., 2007. In situ studies on physiologicaland biochemical responses of four fishes with different trophic levelsto toxic cyanobacterial blooms in a large Chinese lake. Toxicon 3,365–376.

Rabergh, C.M.I., Bylund, G., Eriksson, J.E., 1991. Histophatological effects ofMicrocystin-LR, a cyclic peptide toxicin from the cyanobacterium

M.F. Nince Ferreira et al. / Toxicon 55 (2010) 1255–12621262

(blue-green algae) Microcystis aeruginosa, on common carp (Cyprinuscarpio L.). Aquat. Toxicol. 20, 131–146.

Rao, P.V.L., Bhattacharya, R., 1996. The cyanobactial toxin mirocystin-LRinduced DNA damage in mouse liver in vivo. Toxicology 114, 29–36.

Rao, P.V.L., Bhattacharya, R., Parida, M.M., Jana, A.M., Bhaskar, A., 1998.Freshawater cyanobacterium Microcystis aeruginosa (UTEX 2385)induced DNA damage in vivo and in vitro. Envion. Toxicol. Phar. 5, 1–6.

Rinehart, K.L., Namikoshi, M., Choi, B.W., 1994. Structure and biosynthesisof toxins from blue-green algae (cyanobacteria). J. Appl. Phycol. 6,159–176.

Runnegar, M.T.C., Falconer, I.R., 1986. Effect of toxin from the cyanobac-terium Microcystis aeruginosa on ultrastructural morphology andactin polymerization in isolated hepatocytes. Toxicon 24, 109–115.

Runnegar, M.T.C., Berndt, N., Kong, S.M., Lee, E.Y., Zhang, L., 1995. In vivoand in vitro binding of microcystin to protein phosphatases1 and 2A.Biochem. Bioph. Res. Co. 216, 162–169.

Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J.(Eds.), Toxic Cyanobacteria in Water: a Guide to Their Public HealthConsequences, Monitoring and Management. E & FN Spon, London,pp. 41–111.

Soares, R.M., Magalhaes, V.F., Azevedo, S.M.F.O., 2004. Accumulation anddepuration of microcystins (cyanobacteria hepatotoxins) in Tilapiarendalli (Cichlidae) under laboratory conditions. Aquat. Toxicol. 70,1–10.

Tencalla, G.F., Dietrich, R.D., Schlatter, C.H., 1994. Toxicity of Microcystisaeruginosa peptide toxin to yearling rainbow trout (Oncorhynchusmykiss). Aquat. Toxicol. 30, 215–224.

Toivola, D.M., Eriksson, J.B., 1999. Toxins affecting cell signalling andalteration of cytoskeletal structure. Toxicol. in Vitro. 13, 521–530.

Xie, L.Q., Xie, P., Ozawa, K., Honma, T., Yokoyama, A., Park, H.D., 2004.Dynamics of microcystins-LR and -RR in the planktivorous silver carpin a sub-chronic toxic experiment. Environ. Pollut. 127, 431–439.