Acid and alkaline phosphatase activities and pathological

changes induced in Tilapia fish (Oreochromis sp.) exposed

subchronically to microcystins from toxic cyanobacterial

blooms under laboratory conditions

R. Molinaa, I. Morenoa, S. Pichardoa, A. Josa, R. Moyanob,J.G. Monterdec, A. Cameana,*

aArea de Toxicologıa, Facultad de Farmacia, C/Profesor Garcıa Gonzalez, n8 2, 41012 Sevilla, SpainbDpto. Farmacologıa, Toxicologıa, Medicina Legal y Forense, Campus de Rabanales Carretera Madrid-Cadiz s/n 14071 Cordoba, Spain

cDpto. Anatomıa y Anatomıa Patologica Comparadas. Campus de Rabanales Carretera Madrid-Cadiz s/n. 14071 Cordoba, Spain

Received 3 June 2005; revised 18 July 2005; accepted 20 July 2005

Available online 26 September 2005

Abstract

The effects of microcystins (MCs) from cyanobacterial cells on the enzymatic activities of acid and alkaline phosphatases

(ACP and ALP) from liver, kidney and gill tissues, and the histopathological changes in freshwater Tilapia fish (Oreochromis

sp.) were investigated under laboratory conditions. Fish were exposed to cyanobacterial cells (60.0 mg MC-LR/fish per day)

through their diet at different exposure times (14 and 21 days). The cells were fed to the fish in two types of oral administration:

mixed with a commercial fish food or crushed into a commercial fish food so that the toxins were released. ACP and ALP

activities changed in response to MCs in a time-dependent manner, and these changes were more prominent in liver and kidney.

The way the MCs were administered had no influence on the biochemical parameters. Similarly, the most severe

histopathological changes were observed in the same two organs, although the gills and intestines were also affected. The

parenchymal architecture of the liver was dissolved, and round hepatocytes with the appearance of pyknotic nuclei were

detected. Kidney lesions consisted of the dilation of Bowman’s space and necrotic epithelial cells with pyknotic nuclei in the

tubules. These findings suggest that low and repeated doses of MC-LR from cyanobacterial cells induce toxicity in tilapia fish

although no adverse effects were detected

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Cyanobacteria; Microcystins; Phosphatases; Tilapia; Histopathology

1. Introduction

Cyanobacteria are extremely widespread and occur

throughout the world in fresh and salt water, and various

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2005.07.012

* Corresponding author Address: Bioquımica, Bromatologıa,

Toxicologıa and Medicina Legal, University of Seville, Profesor

Garcıa Gonzalez n8 2, 41012 Sevilla, Spain. Tel.: C34 95 455 6762;

fax: C34 95 423 3765.

E-mail address: camean@us.es (A. Camean).

terrestrial habitats. Several cyanobacteria species, specifi-

cally Microcystis aeruginosa, are capable of producing a

variety of potent toxins, including a group of hepatotox-

ins called microcystins (MCs) which have strong

cytotoxic activity (Codd et al., 1997; de Figuereido

et al., 2004). There are more than 70 MC isoforms

(Fastner, 2002), but the most frequent and most

commonly studied variant is microcystin-LR (MC-LR),

followed by MC-RR, MC-YR and MC-LA (de

Figuereido et al., 2004).

Toxicon 46 (2005) 725–735

www.elsevier.com/locate/toxicon

R. Molina et al. / Toxicon 46 (2005) 725–735726

It is well known that MCs are responsible for illness and

death not only in domestic and wildlife animals but also in

humans (Guzman and Solter, 1999).

In mammalian cells, MCs are selective for hepatocytes.

They irreversibly inhibit protein phosphatases 1 and 2A,

which regulate numerous biological processes, and lead to

an increase in protein phosphorylation (Xu et al., 2000).

Laboratory studies have shown that these toxins increase

reactive oxygen species, induce cytoskeleton disruption,

which results in necrosis and acute hepatotoxicity, and

promote the growth of liver tumours, hepatic neoplastic

nodules and preneoplastic colon tumours (Chernoff et al.,

2002).

Aquatic animals such as zooplankton, fish and molluscs

consumed by humans have been reported to bioaccumulate

MCs (Williams et al., 1997; Amorin and Vasconcelos, 1999;

Freitas de Magalhaes et al., 2003). Cyanobacteria are an

important part of the diet of various tropical fish, including

tilapias (Keshavanath et al., 1994). Actually, Oreochromis

niloticus has been reported to accumulate MCs in gut, liver,

kidney and muscle (Mohamed et al., 2003) like other

freshwater fish (Xie et al., 2005), at levels that are above the

Tolerable Daily Intake (TDI) of 0.04 mg kgK1 of body

weight per dayK1 which the WHO proposed as a provisional

guideline value (Chorus and Bartram, 1999). This is of great

concern for public health because the chronic ingestion of

trace amounts of MCs (MC-LR in particular) in food and

drinking water has considerable potential to promote cancer

(de Figuereido et al., 2004).

Several studies have been performed to assess the impact

of MCs on fish (Bury et al., 1996; Kotac et al., 1996;

Tencalla and Dietrich, 1997; Fischer and Dietrich, 2000;

Fischer et al., 2000; Jacquet et al., 2004), although most of

them have focused on the histopathological changes after

the acute administration of MCs by the intraperitoneal route.

Unlike mammalian studies, fish exposed to MCs suffered

hepatopancreas and kidney damage (Fischer and Dietrich,

2000), and degeneration of the gills (Carbis et al., 1996) and

heart (Best et al., 2001). There are few studies on the cellular

changes induced by the chronic exposure of fish to MCs in

natural contaminated waters (Bury et al., 1995; Carbis et al.,

1997) and under laboratory conditions (Carbis et al., 1996;

Soares et al., 2004; Li et al., 2004; Xie et al., 2004).

Previous studies in our laboratory showed that Tilapia

(Oreochromis sp.) exposed to cyanobacterial cells (60.0 mg

MC-LR/fish per day) under laboratory conditions experi-

enced changes in their endogenous antioxidant defense

system in a time-dependent manner. Simultaneously, the

lipid peroxidation level (LPO) significantly increased in the

liver, kidney and gills of fish exposed for 21 days,

particularly when they were fed with crushed cyanobacterial

cells (Jos et al., 2005).

This paper aims to investigate how lyophilized toxic

cyanobacterial cells affect enzymatic parameters, such as

acid phosphatase (ACP) and alkaline phosphatase (ALP),

and morphological alterations in liver, kidney, gills

and intestinal mucosa (only histopathology) of tilapia fish

(Oreochromis sp.). Tilapia has been chosen as a test

organism because it is an emerging and attractive species

for aquaculture: it grows quickly, is large when it

reproduces, has a low feeding trophic level and is cheap to

produce (Costa-Pierce and Rakocy, 1997). The fish were

exposed to cyanobacterial cells in their diet for two different

lengths of time (14 and 21 days) and two different types of

oral administration were compared. Both enzymes catalysed

the hydrolysis of various phosphate-containing compounds

and acted as transphosphorylases at acid and alkaline pHs,

respectively. They were also involved in a variety of

metabolic processes, such as molecule permeability, growth

and cell differentiation and they have been used as indicators

of intoxication by contaminants (Mazorra et al., 2002).

2. Materials and methods

2.1. Chemicals

MC-LR, -RR and -YR (purity 95–99%) standards were

supplied by Calbiochem-Novabiochem (La Jolla, Ca, USA).

Methanol and ethanol were purchased from Merck

(Darmstadt, Germany) and bovine g-globuline from Biomol

(Plymouth, USA). Mannitol, tricaine methane sulphonate

(MS-222), glutaraldehyde, HEPES, TRIS, and all the other

reagents used were of the highest purity available and were

purchased from Sigma (Steinheim, Germany).

2.2. Animals

Male tilapia fish (Oreochromis sp.) with a mean weight

of 49.92G9.1 g were obtained from a fish hatchery. The fish

were acclimated to laboratory conditions for 15 days before

the experiments were begun, in aquariums (eight indivi-

duals/aquarium) with 96 L of fresh water. They were kept at

a water temperature of 21G2 8C in dechlorinated tap water

under continuous filtration [Eheim Liberty 150 and Bio-

Espumador cartridges (Bio-Espumador)]. Dissolved oxygen

values were maintained between 6.5 and 7.5 mg/L and water

quality was determined for nitrite (nitrite test kit, Aquarium

Pharmaceuticals, Inc., UK) at the beginning of the

experimental period and periodically thereafter. Mean

values for other parameters of water quality were: pH

7.6G0.2, conductivity 292 mS/cm, Ca2C 0.60 mM/L and

Mg2C 0.3 mM/L. Fish were fed once every morning with

commercial fish food.

2.3. Determination of cyanobacterial toxins from

Microcystis waterblooms

Lyophilized cells of cyanobacteria collected from the

river Guadiana (Mertola, Portugal) were kindly supplied by

Dr Susana Franca and Dr Paulo Pereira (Lisbon, Portugal).

In order to determine the MC content of two different

R. Molina et al. / Toxicon 46 (2005) 725–735 727

Microcystis waterblooms, the lyophilized cells were

extracted using the method of Camean et al. (2004).

Quantitative HPLC analysis of the MC levels was

performed using the method of Moreno et al. (2004), on a

250 mm!4.6 mm i.d., 5 mm, LiChrosphere C18 column

purchased from Merck (Darmstadt, Germany). The LC

system used to analyse the toxin contents was a Varian 9012

equipped with a Varian ProStar 330 Diode Array Detector.

Chromatographic data were processed with a Star Chroma-

tography Workstation (Varian Technologies). Standard

solutions of microcystins MC-LR, MC-YR and MC-RR

were prepared in methanol (500 mg/mL) and then diluted as

required with methanol for use as working solutions (0.5–

5.0 mg/L of each toxin). Only MC-LR was identified in the

cyanobacterial cell extracts analysed and the peak area of

the MC-LR standard solution was applied for quantification.

The concentrations of MC-LR obtained from both lyophi-

lized cells were similar, 3230 mg/g MC-LR and 2647 mg/g

MC-LR.

2.4. Experimental exposure and sampling

Tilapia fish were exposed to MC-LR by being fed with

cyanobacterial cells under laboratory conditions for two

different periods of time (14 and 21 days) and two different

types of toxin administration (crushed and non-crushed).

The two experiments designed are described below. A

control group of fish (nZ8) in both experiments was

administered only the commercial fish food for the period

that the exposed groups received food containing MC-LR.

Experiment 1. Fish in a test aquarium (nZ8) were fed

with commercial fish food plus lyophilized cyanobacterial

cells, containing 3230 mg/g MC-LR. M. aeruginosa cells

were fed to the fish by manually crushing a mixture of both

components (fish food and toxic cells) in a mortar. This

mixture was then sonicated. The procedure resulted in small

sticky pellets and was designed to replicate the type of

exposure that may occur when a cyanobacteria bloom

undergoes lysis under field conditions. The pellets were

placed in the tank and drifted to the bottom for the fish to eat.

All the pellets were eaten within an hour. The amount of

commercial fish food administered per fish was 0.3 g/day

and the quantity of cyanobacterial cells was selected so that

each fish consumed approximately 60.0 mg MC-LR/day

(determined by HPLC analysis) for 14 days.

Experiment 2. This experiment was carried out using two

test aquaria with eight fish in each and a control tank. The

fish were exposed to cyanobacterial cells for 21 days. In

accordance with the dose of toxin chosen (approximately

60.0 mg MC-LR/fish per day) and the concentration of MC-

LR contained in the cyanobacterial cells (2647 mg/g MC-

LR), we determined the quantity of lyophilized cells that

had to be added to the aquarium. The lyophilized cells were

administered in two different ways. In one of the test

aquariums, the fish received the toxins in the same way as in

experiment 1 (in pellets containing commercial fish food

plus crushed lyophilized cyanobacterial cells). In the second

test aquarium, the fish were fed with commercial fish food

(0.3 g/fish per day) and lyophilized Microcystis cells (non-

crushed cells) dusted on the water surface, daily for 21 days.

The uptake of lyophilised cells by the fish was complete

within 3 h in both treatments.

Sampling was conducted after both experimental

periods. At sampling, fish were removed at the same time

from each aquarium (control and treated), anaesthetized in

MS-222 before being killed by transaction of the spinal

cord. Liver, kidney, gills and intestines were removed and

weighed. Part of these tissues were rinsed with ice-cold

saline and kept at K85 8C until biochemical analysis. The

rest of the tissue samples were fixed so that the

histopathological study could be carried out.

2.5. Determination of the enzyme activity in liver, kidney

and gills

For the estimation of enzyme activities, liver, kidney and

gill tissues were weighed, homogenised using a Ultra-

Turraxw tissue homogenizer at 4 8C in a buffer containing

100 mmol/L mannitol, 2 mmol/L HEPES/TRIS, pH 7.1,

0.1 mmol/L benzamidine and 0.2 mmol/phenylmethyl-

sulfonyl fluoride. The use of acid phosphatase (ACP) as a

marker enzyme for lysosomal membranes and alkaline

phosphatase (ALP) as an apical membrane enzyme was

analysed with the methods of Pennington (1961); Bretau-

diere et al. (1977), respectively. The protein content of the

tissues was quantified with Bradford’s method (1976) and

with bovine g-globuline as the standard. The optical density

was measured spectrophotometrically at 400 nm (ALP) and

405 nm (ACP), using a U-2001 Hitachi spectrophotometer

(USA). Enzyme activity was expressed as nmol/mg protein

per minute.

2.6. Light microscopy and electron microscopy

Tissue samples were taken for histological examination

from the liver, kidney, gills and intestines of the control and

exposed fish on the 21-day crushed diet (experiment 2). For

light microscopy, samples were first fixed in 10% buffered

formalin for 24 h at 4 8C, and then immediately dehydrated

in graded series of ethanol, immersed in xylol and

embedded in paraffin wax using an automatic processor.

Sections of 3–5 mm were mounted. After they had been

deparaffinized, the sections were rehydrated, stained with

hematoxylin and eosin, mounted with Cristal/Mount and

subsequently subject to pathological assessment.

Liver tissue sections were also stained with periodic acid

Schiff (PAS) so that the glycogen content of the hepatocytes

could be evaluated.

For electron microscopy, samples were prefixed in 2%

glutaraldehyde fixative (in pH 7.4 phosphate buffer for 10 h

at 4 8C) and postfixed in 1% osmium tetroxide fixative (in

pH 7.4 phosphate buffer for 0.5 h at 4 8C). Subsequently,

R. Molina et al. / Toxicon 46 (2005) 725–735728

they were dehydrated in graded ethanol series and

embedded in epon. Ultra thin sections, 50.60 nm, were cut

with a LKB microtome. The sections were mounted on

copper grig and stained with uranylacetate and lead citrate.

The tissue sections were examined on a Philips CM10

electron microscope.

2.7. Statistical analysis

Results are presented as the meanGSE. The differences

between the data from the various types of cyanobacterial

cell administration for 21 days were analyzed by one-way

analysis of variance (ANOVA). Then, an unpaired two-

tailed Student’s t-test was used and differences were

considered to be significant at p!0.05. All other compari-

sons were performed by the unpaired t-test.

3. Results

3.1. General characteristics in experimental groups

No fish died during the exposure periods considered (14

or 21 days). The behavior of the MC-treated fish could not

be distinguished from that of the controls in any of the

experiments.

Table 1 shows that the relative weights of liver, kidney

and gills of the fish exposed for 14 and 21 days were not

affected. Protein content, expressed as mg protein/g organ,

decreased in the liver of exposed fish in a time-dependent

manner; likewise, this significant effect was also observed in

the gills of exposed fish. In contrast, no changes were found

in kidney in any of the exposed groups.

3.2. Effect of cyanobacterial cells on ALP and ACP

enzymatic activities

ACP activity increased significantly in the liver and

kidney of fish exposed to cyanobacterial cells for 14

Table 1

General characteristics of control and exposed fish to lyophilised cyanoba

Organ Control 14 days

Liver

g/10 g b.w. 0.086G0.010 0.099G0.015

mg prot/g liver 116.56G42.59 105.41G26.55

Kidney

g/10 g b.w. 0.018G0.002 0.018G0.003

mg prot/g kidney 140.25G26.11 138.57G6.80

Gills

g/10 g b.w. 0.293G0.050 0.311G0.030

mg prot/g gills 0.30G0.01 0.25G0.02**

p!0.05 compared with control group. p!0.01 compared with control gr

and 21 days (p!0.001, both diets), whereas in gills no

change was appreciated (Fig. 1).

ALP activity showed no significant differences in the

liver of fish exposed to crushed cyanobacterial cells for

14 days; after 21 days, however, the increase was significant

(p!0.001, both diets) (Fig. 2). The effect of cyanobacterial

cells on this parameter was more pronounced in the kidney

of exposed fish, and differences were significant even after

14 days of exposure. Results were similar after 21 days of

exposure, and levels of ALP activity were higher when the

cyanobacterial cells were crushed with the fish food. Finally,

no significant differences were observed in ALP levels in

gills, although this enzyme was more active in fish after

14 days of treatment (13%). After the longer exposure

(21 days), the percentages of activity were higher,

particularly with the crushed diet.

3.3. Histopathological study

3.3.1. Macroscopical observations

No gross pathological changes were observed in fish

treated with cyanobacterial cells for 14 days in any of the

organs studied, the appearance of which was macroscopi-

cally normal, just like that of the fish in the control groups.

The livers from the fish treated with cyanobacterial cells

for 21 days (crushed and non-crushed) presented yellowish

discolorations unlike the control livers, which were dark red.

No overt gross pathological changes were observed in any

of the other organs.

3.3.2. Light microscopy

Although the histological samples from the control

groups had the expected normal appearance (Figs. 3A, E,

4A, and 5A, C), the organs studied from the treated fish

underwent structural alterations, the severity of which

increased with the period of exposure (14 and 21 days).

There were no differences between the types of adminis-

tration (crushed and non-crushed).

The livers from the treated fish underwent structural

alterations; the cord-like parenchymal architecture of

cterial cells

21 days

Crushed Non-crushed

0.097G0.019 0.105G0.023

39.73G12.82*** 48.23G7.19***

0.021G0.006 0.016G0.006

138.30G21.00 140.80G23.33

0.300G0.040 0.320G0.020* 0.18G0.02*** 0.20G0.02***

oup. ***p!0.001 compared with control group.

Fig. 1. Acid phosphatase activity (ACP) in liver, kidney and gills of

control fish and fish exposed to cyanobacterial cells (60.0 mg/fish

per day) following two different types of administration: crushed

and non-crushed food. The values are expressed as meanGSE (nZ8).

The significance levels observed are ***p!0.001 in comparison to

control group values.

Fig. 2. Alkaline phosphatase activity (ALP) in liver, kidney and gills

of control fish and fish exposed to cyanobacterial cells (60.0 mg/fish

per day) following two different types of administration: crushed

and non-crushed food. The values are expressed as meanGSE (nZ8). The significance levels observed are **p!0.01 and ***p!0.001 in comparison to control group values.

R. Molina et al. / Toxicon 46 (2005) 725–735 729

the liver was dissolved, primarily in the perilobular regions

of the liver (14-day group), and over time this became more

pronounced and involved larger areas of the liver in the fish

treated with cyanobacterial cells (21-day group). The

histopathological changes in the hepatocytes were charac-

terized by condensed PAS negative hyaline cytoplasms

associated in many cases with the appearance of pyknotic

nuclei and such other signs of cellular necrosis as

cytoplasmatic vacuolation and shrinkage (Fig. 3B and C).

In the pancreatic acinus, many lipid vacuoles and necrotic

cells were observed (Fig. 3D and F).

Kidney lesions ranged from dilation of the Bowman

capsule and vacuolation of the tubular lining (after 14 days

of treatment) to evidence of toxic nephrosis (after 21 days of

treatment) characterized by necrotic epithelial cells with

pycnotic nuclei (Fig. 4B). Other evidence of nephrosis such

as generalized urolith-like basophilic precipitate in the

tubules was also observed (Fig. 4C).

Histopathological changes in the gills were detected

only in the fish treated with cyanobacterial cells for 21 days

and consisted of generalized hyperaemia with the epi-

thelium separated from the underlying vascular spaces

(Fig. 5B).

Intestinal pathological changes were also observed after

21 days of treatment. These changes were characterized by

hyperplasia and pyknotic cells lining the intestinal mucosa

(Fig. 5D).

3.3.3. Ultrastructural observations

In the 14-day treated group, the cytoplasms of

hepatocytes were highly vacuolated. They contained lipids

and proteins, and lost glycogen; mitochondria were

dispersed in the cytoplasm and slightly swollen. After

21 days of treatment, the dissociation of the hepatocytes was

more evident, and the RER was highly vesiculated.

Numerous necrotic nuclei and autophagocytotic vesicles

containing remnants of organelles were also observed

(Fig. 6A).

The proximal tubules in the kidney of exposed fish

contained more lysosomes, and a considerable pleomorphic,

generalized vacuolization of the tubular epithelial cells was

also detected (Fig. 6B). Necrotic tubular epithelial cells

were often observed adjacent to seemingly healthy tubules.

Fig. 3. Effects of MC-LR from cyanobacterial cells on the histology of exposed tilapia hepatopancreas (60 mg MC-LR/fish per day). (A) Liver

parenchyma of control tilapia fish. (B) Alteration in the structural organization of hepatocytes and necrosis (*). (C) Hepatocytes showing

hyaline condensed cytoplasms PAS negative (*). (D) Hepatopancreas showing lipid vacuoles (/) and necrotic cells (*). (E) Pancreatic acinus

of control tilapia fish. (F) Pancreatic acinus showing lipid vacuoles (/) and necrotic cells (*). Bars represent 20 mm.

R. Molina et al. / Toxicon 46 (2005) 725–735730

4. Discussion

The present study shows that microcystin-LR (MC-LR)

contained in lyophilized cyanobacterial cells induces

changes in some hepatic and renal enzymes (ACP and ALP

activities) and damages the hepatopancreas, kidney, gills and

gastrointestinal tract in tilapia fish (Oreochromis sp.)

exposed to repeated doses of toxin, under laboratory

conditions. The enzyme activities in the vital organs of the

fish were altered and histopathological changes were also

induced, although there were no observable effects in this

species.

Very few studies have been performed on the subchronic

oral toxicity of MCs in fish under laboratory conditions, but

the accumulation of MCs in Tilapia rendalli (Soares et al.,

2004) and silver carp (Xie et al., 2004) have been reported.

Li et al. (2004) showed growth inhibition and severe damage

to hepatocytes in MC-treated carps. In a previous study, we

demonstrated that oxidative stress plays an important role in

in vivo MC-LR induced toxicity in tilapia fish (Jos et al.,

2005).

In the present study, the activities of the membrane

enzymes acid phosphatase (ACP) and alkaline phosphatase

(ALP) in vital organs (liver, kidney and gills) were

evaluated and the capacity of MC-LR to interfere with

metabolic processes studied.

As previously observed in rats treated with MC-LR

(Moreno et al., 2003), the protein content of the liver

Fig. 4. Effects of MC-LR from cyanobacterial cells on the histology of tilapia kidney, exposed for 21 days (60 mg MC-LR/fish per day). (A)

Kidney of control tilapia fish. (B) Necrotic epithelial cells (*) and pyknotic nuclei in the tubules (%). Dilated Bowman’s space in the glomeruli

(/). (C) Nephrosis (*) characterized by severe generalized precipitate basophilic in the tubules like urolithes (/). Bars represent 20 mm.

R. Molina et al. / Toxicon 46 (2005) 725–735 731

and gills of tilapia fish exposed to lyophilized cyanobacter-

ial cells decreased in comparison to control fish after both

exposure times (14 and 21 days). A concentration-

dependent reduction in the protein content was also

observed in vitro, when two fish cell lines (PLHC-1 and

RTG-2) were acutely exposed to MC-LR and MC-RR

(Pichardo et al., 2005). This could be due to the proteolysis

and retardation of protein synthesis produced by MCs.

Fig. 5. Histology of gills and intestinal mucosa from tilapia exposed for 21 days to MC-LR from cyanobacterial cells (60 mg MC-LR/fish per

day). (A) Gills of control tilapia fish. (B) Generalized hyperaemia in gills (*). (C) Intestinal mucosa of control tilapia fish. (D) Hyperplasia (*)

and pyknotic cells (/) lining the intestinal mucosa. Bars represent 20 mm.

R. Molina et al. / Toxicon 46 (2005) 725–735732

This hypothesis is in concordance with the reduction in

protein content observed in muscle and serum of carps

exposed to other pollutants as pyretroids (Das et al., 2002).

The increase in ACP activity observed in the liver and

kidney of exposed fish agrees with previous investigations

carried out on the intestinal homogenates of rats that had

been intraperitoneally exposed to 100 mg/kg MC-LR

(Moreno et al., 2003). Gills showed no statistical change

in this parameter. As previously observed in the liver

Fig. 6. Ultrastructural effects of MC-LR on liver and kidney of tilapia expo

pyknotic nuclei (*) and cytoplasm highly vacuolized containing lipids and

proximal tubules (*). Generalized vacuolization in kidney (%).

and muscle of fish and prawn hepatopancreas, because of

exposure to environmental stressors MCs increase ACP

activity in both organs by interacting with lysosomes

(Ghorpade et al., 2002; Bhavan and Geraldine, 2004). This

agrees with the ultrastructural changes obtained in our study

and also with previous in vitro investigations carried out on

fish cell lines (RTG-2 and PHLC-1), which found a very

potent concentration-dependent stimulation of the lysoso-

mal function with MC-LR (Pichardo et al., 2005). This may

sed for 21 days (60 mg MC-LR/fish per day). (A) Hepatocytes with

proteins, and loss of glycogen (%). (B) Increase of lysosomes in the

R. Molina et al. / Toxicon 46 (2005) 725–735 733

be related to the cytoskeletal modifications and the

induction of oxidative stress that have been pointed out by

several authors (Ding and Ong, 2003; Bouaicha and

Maatouk, 2004; Moreno et al., 2005a).

ALP activity increased significantly in the liver of MC-

LR-exposed tilapia only after 21 days of exposure, and the

changes were more pronounced in kidney. In higher

animals, ALP activity is involved in bone formation and

in membrane transport activities. In the blue crab,

Callinectes sapidus, ALP activity modulates the osmor-

egulatory response (Lovett et al., 1994). ALP is basically a

membrane-bound enzyme, and any perturbation in the

membrane properties caused by interaction with MCs could

alterate the ALP activity. The data show that the MC-LR

contained in cyanobacterial blooms is capable of altering

ALP activity in a time-dependent manner in the liver,

kidney and gills (not significantly in the latter case) of

exposed tilapia. This result is in agreement with previous

studies reporting ALP changes due to the accumulation of

contaminants in fish (Karuppasamy, 2000). The present

study is the first to focus on microcystins.

In contrast, ACP and ALP activities in gills were not

significant. In fish, gills are the first organ targeted by

several xenobiotics because they have a very large interface

area between the external and internal fish environment, and

changes in gill epithelia have been considered to be good

indicators of the effects of toxicants (Jiraungkoorskul et al.,

2003). The literature shows that ACP and ALP activity

decreases in the gills of fish exposed to various substances

(Karuppasamy, 2000; Bhavan and Geraldine, 2004; Das et

al., 2004), but none of the reports focused on the effects of

MCs. Gills were less affected in our study probably because

the fish were exposed orally to the MCs.

The way in which the microcystins were administered—

that is to say, whether the cyanobacterial cells were crushed

or not in the commercial fish food—had no influence on the

increases observed in these enzymes.

The literature that deals with the histopathological

effects of MCs on fish revealed that toxic cyanobacterial

blooms can be associated with fish mortality (Zimba et al.,

2001). In general, the gross histopathological and ultra-

structural changes in various fish species after acute

exposure to either M. aeruginosa and/or purified MC

analogs are similar in many respects to those seen in

mammals. However, there are also several differences

(Zurawell et al., 2005). In addition to severe damage and

dysfunction of the liver, MCs also induce pathological

changes in kidney (Fischer and Dietrich, 2000), and

degeneration in gills (Carbis et al., 1997) and heart (Best

et al., 2001).

Some laboratory studies and field observations suggest

that oral and immersion exposure of fish to MCs is slow to

induce adverse effects and mortality, so acute toxic episodes

are generally quite rare (Zurawell et al., 2005). However, a

few studies on the subchronic oral toxicity of MCs in fish

demonstrate that low concentrations of these toxins cause

hepatopancreas and kidney damage in European carp

(Fischer and Dietrich, 2000) and hepatotoxicosis and liver

necrosis in the rainbow trout (Fischer et al., 2000).

Among our histological findings were a loss of the

parenchymal architecture, necrosis of the hepatocytes, and

an increase in the size of lipid droplets in the pancreatic

acinus. In the PAS staining of liver sections, all fish exposed

to MC-LR had a lower glycogen content in the liver than the

control fish. The vacuolization of hepatocytes might

indicate an imbalance between the rate of synthesis of

substances in the parenchymal cells and the rate of their

release into the systemic circulation. This seems to confirm

that MCs are liver specific toxins and is similar to what

happened when carps were injected with sublethal doses of

MC-LR (Rabergh et al., 1991) or gavaged with a single

sublethal bolus dose of M. aeruginosa cells (Fischer and

Dietrich, 2000). In the latter case, apoptosis was the primary

mechanism of cell death.

The Bowman space in the kidney of exposed tilapia

dilated, the number of lysosomes in the proximal tubules

increased and there were many vacuolaes. These results are

consistent with those obtained from carps that were

immersed in tanks containing MCs from cyanobacterial

cells (Carbis et al., 1996) or gavaged with a single sublethal

bolus dose of MC-LR (Fischer and Dietrich, 2000). MC-LR

has been proved to promote renal alterations and affect renal

physiology (Nobre et al., 2003) because the kidney is

involved in eliminating MC-LR from the body.

Finally, the gills and the gastrointestinal tract were the

least affected organs. In fact, branchial injury is most severe

when microcystins are administered via the peritoneal route

and the damage only occurred when the fish were immersed

in a toxic cell-free lysate of M. aeruginosa containing

soluble MCs (Carbis et al., 1996) or after the lysis of a

cyanobacterial bloom (Rodger et al., 1994). This may be

because delayed or subchronic branchial damage in fish

might occur after microcystins have been taken up by the

intestines. In this respect, previous studies made in our

laboratory have demonstrated that MCs accumulate in the

liver and intestines of tilapia exposed to repeated doses of

MC-LR from cyanobacterial cells (Moreno et al., 2005b).

In summary, the results of this study show that when

tilapia were exposed to cyanobacterial cells under labora-

tory conditions (60.0 mg MC-LR/fish per day) the enzymatic

activities of acid and alkaline phosphatases (ACP and ALP)

changed in a time-dependent manner, but adapted to the

toxic environment over time. At this concentration, MC-LR

is only moderately toxic in tilapia fish (Oreochromis sp.),

especially in liver and kidney, and they can derive energy

from alternative pathways and survive. In addition, both

organs underwent histopathological changes, which could

be correlated to the significant increases in ACP and ALP;

the gills and gastrointestinal tract were also affected. These

findings suggest that low and repeated doses of MC-LR

from cyanobacterial cells induce toxicity in tilapia fish

although no adverse effects were perceived. Because these

R. Molina et al. / Toxicon 46 (2005) 725–735734

organisms are an important food source for birds, fish, and

mammals (including humans), MCs in fish and other aquatic

animals should be monitored so that any potential risk can

be evaluated and potential enzyme activities as indicators of

intoxication can be found.

Acknowledgements

The authors wish to thank the CICYT (AGL 2002-

02622) for the financial support for this study.

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