Comparative toxicity evaluation of cyanobacterial cyclicpeptide toxin microcystin variants (LR, RR, YR) in mice

Nidhi Gupta, S.C. Pant, R. Vijayaraghavan, P.V. Lakshmana Rao *

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India

Received 8 January 2003; received in revised form 10 March 2003; accepted 10 March 2003

Abstract

The cyclic peptide toxins microcystins and nodularins are the most common and abundant cyanotoxins present in

diverse water systems. They have been the cause of human and animal health hazards and even death. Over 60

microcystin variants have been reported so far. We report here the results of our study on comparative toxicity

evaluation of three most predominant microcystins, MC-LR, MC-RR and MC-YR in mice. The mice were

administered one LD50 dose of MC-LR, RR and YR (43, 235.4 and 110.6 mg/kg body weight, respectively), and

biochemical and histological variables were determined at 30 min post-treatment and mean time to death (MTD).

Significant increase in liver body weight index was induced by all three variants. There was marginal increase in serum

levels of hepatic enzymes viz. AST, ALT and g-GT at 30 min post-treatment but 3�/4 fold increase was observed at

MTD. In contrast, enhanced LDH leakage, DNA fragmentation and depletion of hepatic glutathione was observed at

30 min post treatment in all three variants. There was no change in levels of serum protein, albumin and albumin/

globulin ratio. Liver histology showed time dependent severe pathological lesions like congestion, haemorrhage, portal

mononuclear cell infiltration and obliteration of chromatin material. Lung lesions were predominantly in bronchi and

parenchyma. Though qualitatively lesions were identical in all three microcystin variants, degree of liver and lung

lesions varied quantitatively with the toxin. The breathing pattern and respiratory frequency of the mice after i.p.

administration of the toxin showed uniform pattern for 90 min followed by abrupt change in the respiratory pattern

and instantaneous death. Based on biochemical and histological studies, MC-LR was found to be the most potent toxin

followed by MC-YR and MC-RR.

# 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Cyclic peptide; Cyanotoxins; Microcystin-LR; Microcystin-RR; Microcystin-YR; Microcystin variants; Hepatotoxin

1. Introduction

The occurrence of toxic freshwater blooms of

cyanobacteria (blue�/green algae) has been re-

ported in many countries. Toxic cyanobacteria

found in eutrophic, freshwater, municipal and

residential water supplies are increasing environ-

* Corresponding author. Tel.: �/91-751-234-1980; fax: �/91-

751-234-1148.

E-mail address: pvlrao@rediffmail.com (P.V.L. Rao).

Toxicology 188 (2003) 285�/296

www.elsevier.com/locate/toxicol

0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.

doi:10.1016/S0300-483X(03)00112-4

mental hazard in several parts of the world (Coddet al., 1989; Dawson, 1998). The toxins of fresh-

water cyanobacteria are classified into two groups,

neurotoxins and hepatotoxins, which include cyc-

lic-peptide microcystins and nodularin. Microcys-

tins and related polypeptides are selectively

hepatotoxic to fish, birds and mammals. The

consequence of acute poisoning by these com-

pounds is rapid disorganization of the hepaticarchitecture, breakdown of sinusoidal structures

and, in mammals, pooling of blood in the liver

(Carmichael, 1992; Rao et al., 1995). Chronic

uptake of microcystins results in generalised he-

patocyte degeneration with necrosis, progressive

fibrosis and mononuclear leukocyte infiltration.

Human illnesses attributed to cyanobacterial tox-

ins can be categorised into gastroenteritis andrelated diseases, allergic and irritation reactions,

and liver diseases (Bell and Codd, 1994; Hitzfeld et

al., 2000; Chorus et al., 2000). Micrcoystins and

nodularin have been found to be potent inhibitors

of protein phosphatase type 1 and 2A as well as

skin and liver tumour promoters in laboratory

animals (Nishiwaki-Matsushima et al., 1992).

They are also suspected to be involved withpromotion of primary liver cancer in humans

exposed to long-term doses of these peptide toxins

through drinking water (Yu, 1995). The bioaccu-

mulation of cyanotoxins by aquatic animals in-

cluding fish, mollusc and zooplankton has been

reported (Amorin and Vasconcelos, 1999), which

indicates that oral consumption by animal tissues

containing cyanotoxins is possible and can lead tohuman toxicity. Tragic death of 60 haemodialysis

patients was reported in Brazil due to the presence

of cyanobacterial toxins in the water supply used

in haemodialysis unit (Azevedo et al., 2002).

Over 60 variants of microcystin have been

reported so far which conform to the following

generalised format: Cyclo (D-Ala1-X2-D-MeAsp3-

Y4-Adda-Arg5-D-Glu6-Mdha7-) where, X and Yare variable amino acids, D-MeAsp is D-erythro-b-

methylaspartic acid, Adda is (2S, 3S, 8S, 9S)-3-

amino-9-methoxy-2-6-8-trimethyl 10-phenyldeca-

4.6-dienoic acid and Mdha is N -methyldehydroa-

lanine. The unusual amino acid Adda is essential

for expression of biological activity. Combinations

of the two variable L-amino acids, X and Y,

account for many of the microcystin variants andare used in the nomenclature of the toxins. The

XY variable amino acids for MC-LR, MC-RR

and MC-YR are leucine (L), arginine (R) and

tyrosine (Y). There is very little information on the

toxicity profile of microcystin variants other than

MC-LR. The objective of the present study was

comparative evaluation of biochemical and histo-

logical effects of three commonly occurring micro-cystin variants, viz. microcystin-LR, RR and YR

in mice.

2. Materials and methods

2.1. Chemicals

The cyanobacterial toxin microcystin-LR, RR

and YR were obtained from Professor W.W.

Carmichael, Wright State University, OH, USA.

All other chemicals were obtained from Sigma

Chemical Co. (St Louis, USA) unless otherwise

specified.

2.2. Animals

Swiss albino female mice weighing between 24

and 26 g body weight from Establishment’s animal

facility were used for the study. The animals were

housed in polypropylene cages with dust-free rice

husk as bedding material, and were provided with

pellet food (Amrut Laboratory Feeds, Maharash-

tra, India) and water ad libitum.

2.3. Experimental protocol

Microcystin variants LR, RR and YR were

dissolved in minimum amount of methanol (0.1%)

and diluted to required concentration in PBS. The

mice were housed four to a cage and allowed to

acclimatise 7 days prior to dosing. Acute 24 h,

LD50 was determined for all the toxins by Dixon’sup and down method for small samples (Dixon,

1965). Animals were divided into four groups of

four animals each and administered as below:

Group Treatment

I PBS (0.2 ml)

N. Gupta et al. / Toxicology 188 (2003) 285�/296286

II One LD50 MC-LRIII One LD50 MC-RR

IV One LD50 MC-YR

All the experiments were repeated at least two

times. The study has the approval of Institute’s

ethical committee on animal experimentation.

2.4. Biochemical parameters

Blood was collected from retro-orbital plexus of

mice at 30 min and at mean time to death (MTD).

Serum harvested from each mice at specified time

points was used to determine serum activity of

lactate dehdydrogenase (LDH), g-glutamyl trans-

peptidase (g-GT) by commercial diagnostic kits of

E. Merck India Ltd. LDH and (g-GT) were

estimated by kinetic method using NADH and g-glutamyl-p -nitroanilide, respectively, as substrates

at 340 nm (LDH) and 405 (g-GT) using Milton

Roy 1201 Spectrophotometer. Serum albumin

concentrations and total protein were measured

and the albumin/globulin (A/G) ratio was deter-

mined. Soon after blood sampling at 30 min and

MTD animals were sacrificed by cervical disloca-

tion, and liver and lung were excised quickly. Liversamples were washed free of adhering extraneous

material, blotted and weighed to determine organ/

body weight index (OBI�/organ weight�/100/

body weight). Protein was estimated by Lowry’s

method (Lowry et al., 1951). GSH was estimated

fluorimetrically using o -pthaladehyde (OPT) as a

reagent (Hisin and Hilf, 1976) in a Shimadzu RF

5000 spectro-fluorophotometer.

2.5. DNA fragmentation assay

DNA fragmentation was assayed as previously

described (Rao et al., 1999). Briefly, the liver from

control and treated animals were quickly excised

and frozen. The frozen tissues were homogenised

in ice-cold lysis buffer (10 mM Tris, 20 mM

EDTA, 0.5% Triton X-100, pH 8.0) and thencentrifuged at 27 000�/g for 30 min. Both pellet

(intact chromatin) and supernatant (DNA frag-

ments) were assayed for DNA content fluorime-

trically by using fluorescent dye 4?,6-diamidino-2-

phenylindole (DAPI). Briefly, to 2 ml of the

reagent 100 ng/ml NaCl, 20 ml of a sample were

added and then fluorescence intensity was mea-sured at 450 nm with excitation at 362 nm. The

percentage of fragmented DNA was defined as the

ratio of the DNA content of the supernatant at

27 000�/g to the total DNA in the lysate (Wyllie,

1980).

2.6. Measurement of respiratory variables

For each toxin four animals were used. They

were restrained in body plethysmograph for the

recording of respiratory signals. A volumetric

pressure transducer (model PT5, Grass Instru-

ment, USA) was used for sensing respiratory

flow signals. A continuous airflow of 170 ml/min

was maintained into each body plethysmograph

using a critical orifice (27 gauge needle). Thesignals from the individual transducer were am-

plified using universal amplifiers (Gould, USA).

The amplified signals were digitised using an

analogue to digital converter (Metrabyte, Taun-

ton, USA) and stored and analysed in a personal

computer. The amplified signals from the universal

amplifier were also fed into an oscillograph for

recording of breathing pattern (WindoGraf,Gould, USA). The animals were acclimatised in

the body plethysmograph for 30 min. After the

acclimatization a control recording of respiratory

variables was carried out for 30 min. One LD50

dose of the toxins MC-LR, MC-RR or MC-YR

was administered intraperitoneally after 30 min.

The respiratory variables were recorded for a

period of 4 h after administration. All the respira-tory variables, viz. tidal volume (VT), inspiratory

time (TI), expiratory time (TE) and respiratory

frequency (f) were measured. A computer program

capable of recognising the effects of chemicals as

sensory irritation, airway obstruction or pulmon-

ary irritation, and a combination of these effects,

was used for the collection and analysis (Vijayar-

aghavan et al., 1994).

2.7. Histology

Mice were treated with one LD50 dose of the

toxin. The animals were sacrificed by cervical

dislocation at specified time points (30 min and

MTD). Tissue samples of liver and lung were

N. Gupta et al. / Toxicology 188 (2003) 285�/296 287

obtained from both control and treated animals.Complete necropsy was performed in tissues from

liver and lung. For pulmonary tissue, trachea was

tied before opening the thoracic cage, lung with

the trachea was excised and fixed in 10% buffered

formalin solution. After proper fixation, small

pieces of lung and liver were processed by dehy-

dration and embedded in paraffin. Multiple sec-

tions from each block were prepared at 5�/6 mmthickness and stained with haematoxylin and eosin

for examination under light microscope. The

severity of the lesions were characterised and

scored using Leica Q Win-500 1W image analyser.

2.8. Statistical analysis

Data were expressed as mean9/S.E. from four

animals per each treatment. Data were analysed by

one-way Analysis of Variance (ANOVA) followed

by multiple mean comparison by Student�/New-

man�/Keul’s test. The level of significance was set

at P 5/0.05.

3. Results

The acute 24 h LD50 (i.p.) of three microcystins

was determined by Dixon’s up and down methodfor small samples to minimise the number of

animals (Dixon, 1965). Table 1 summarises the

molecular formulae and LD50 of the three toxins.

MC-LR was most toxic followed by MC-YR and

MC-RR. The time to death varied considerably. In

MC-LR and MC-YR, MTD was similar com-

pared with higher survival time of MC-RR. For all

subsequent studies on biochemical and histologicalparameters animals were sacrificed at 30 min post

treatment and at MTD when animals were mor-

ibund (referred hereafter as MTD) in order to

examine immediate and delayed effects of the

toxin. Clinical signs of toxicity observed within 1

h of treatment included restlessness, laboured

ventilation, in coordination of movement, spas-modic leaping and splaying of hind limbs. Death

was preceded by severe convulsions. Upon ne-

cropsy the animals showed grossly distended liver

engorged with blood. The liver body weight index

(LBI) of the mice treated with three microcystin

variants at 30 min and before death is shown in

Table 2. At 30 min post-treatment there is no

significant increase in LBI compared with control.However, all three toxins showed significant

increase in LBI at MTD compared with control

group.

The serum biochemical variables measured in-

clude alanine amino transferase (ALT), aspartate

amino transferase (AST), g-glutamyl transpepti-

dase (g-GT), total protein, albumin, globulin,

albumin/globulin ratio (A/G). In addition, hepaticglutathione content (GSH) and hepatic DNA

Table 1

The chemical formulae of microcystin variants, their LD50 values and mean survival time in mice

Toxin Formula MW (mg/kg, i.p.) LD50 Fiducial limits MTD (min)a

MC-LR C49H74N10O12 994 43.0 (37.5�/49.4) 97.59/12.9

MC-RR C49H75N13O12 1037 235.4 (202.3�/272.8) 158.79/9.6

MC-YR C52H72N10O13 1044 110.6 (81.7�/149.6) 76.29/9.8

a Values are mean9/S.E. of four animals for each treatment.

Table 2

Liver body weight index of mice treated with LD50 dose of

microcystin-LR, RR and YR

Toxin Dose (mg/kg) Liver body weight index*

30 min MTD

Control �/ 5.569/0.09a 5.569/0.09a

MC-LR 43.0 6.979/0.47a 7.299/0.12b

MC-RR 235.4 5.799/0.47a 7.259/0.50b

MC-YR 110.6 5.649/0.71a 7.549/0.25b

Values are mean9/S.E. of four animals per treatment.

* Means followed by different alphabet are significantly

different at P 5/0.05 by Student�/Newman�/Keul’s test.

N. Gupta et al. / Toxicology 188 (2003) 285�/296288

fragmentation were estimated. All these biochem-

ical variables were determined at 30 min post-

treatment and MTD. Effect of microcystin var-

iants on plasma enzymes of hepatic origin AST,

ALT and g-GT are shown in Fig. 1A�/C. At 30

min post-treatment there was significant change in

levels of these enzymes from control group. But at

MTD a further significant increase in the levels of

AST, ALT and g-GT was observed. There was no

significant difference among the three variants at

30 min post-treatment for the three enzymes AST,

ALT and g-GT. At MTD, MC-LR induced

significant increase in AST compared with MC-

RR and MC-YR. Similar increased leakage was

observed with respect to ALT and g-GT at MTD

in MC-LR group than MC-RR (Fig. 1B, C). There

is no significant difference in LDH leakage at 30

min among the three variants but more than 2-fold

Fig. 1. Effect of microcystin variants LR, RR and YR on

serum levels of (A) AST, (B) ALT, and (C) g-GT after i.p.

administration of one LD50 dose of toxin. Values are mean9/

S.E. of four animals each. * Means followed by different

alphabet(s) are significantly different at P 5/0.05 by Student�/

Newman�/Keul’s test.

Fig. 2. Effect of microcystin variants LR, RR and YR on (A)

LDH leakage, (B) hepatic GSH content, and (C) hepatic DNA

fragmentation after i.p. administration of one LD50 dose of

toxin. Values are mean9/S.E. of four animals each. * Means

followed by different alphabet(s) are significantly different at

P B/0.05 by Student�/Newman�/Keul’s test.

N. Gupta et al. / Toxicology 188 (2003) 285�/296 289

increase was observed at MTD (Fig. 2A). Fig. 2B

shows the hepatic GSH content after microcystin

administration. There was significant decrease in

GSH content 30 min post-treatment with respect

to control group. At MTD, decrease in GSH

content was significant in MC-LR group com-

pared with MC-RR and MC-YR. The hepatic

DNA fragmentation was observed as early as 30

min post-treatment and increased further at MTD

(Fig. 2C). DNA fragmentation was more in MC-

LR treated group than MC-RR and MC-YR at 30

min post-treatment but at MTD there is nodifference among the three variants. There was

no significant change in profile of serum total

protein, albumin and A/G ratio of all three

microcystin variants at both time points (Fig.

3A, B).

Liver histology of control mice showed normal

hepatic cord pattern, hepatic lobules and hepato-

cytes (Fig. 4A). Microcystin exposure causedsevere pathological lesions in hepatic tissues. Liver

histology showed fatty granular degeneration of

varying grades with increase at post-exposure

period. At 30 min post-treatment, severity of

lesions were mild to moderate. At MTD, hepatic

degeneration became more pronounced. Severely

lesioned liver showed congestion, haemorrhage,

portal mononuclear cell infiltration and oblitera-tion of chromatin material (Fig. 4B�/D). These

lesions were evidenced by accumulation of eosino-

philic debris and degenerated cellular components.

A summary of hepatic lesions is shown in Table 3.

The lung histology of control mice is shown in Fig.

4E. The airways, air sacs and alveolar septa were

normal. The pulmonary parenchyma of the

bronchi were intact and showing normal histology.Histological examination of mice lung following

exposure of different variants of microcystins

revealed that the lesions were predominantly in

the bronchi and parenchyma. It was qualitatively

identical in all the variants and varied quantita-

tively in the intensity of lesions. The bronchial

parenchyma was largely intact, displaying focal

degeneration and sloughing off epithelial cells.Pulmonary inflammation, congestion and haemor-

rhage were significant observations. At 90 min

post-treatment, the injured bronchial epithelium,

parenchyma and cellular debris were admixed with

haemorrhagic and fibrinoid material creating fi-

brinocellular network (Fig. 4F�/H). At this point

severe congestion and haemorrhage was also

observed. A summary of pulmonary lesions isgiven in Table 4.

The breathing pattern and the respiratory vari-

ables, viz. VT, TI, TE and F, of the mice after i.p.

administration of the toxins showed abrupt change

in the respiratory variables and died instanta-

neously. Out of four mice administered MC-LR,

two died within 4 h observation period. In MC-

Fig. 3. Effect of microcystin variants LR, RR and YR on

serum total protein, albumin and A/G ratio after (A) 30 min

post-treatment, and (B) MTD after i.p. administration of one

LD50 dose of toxin. Values are mean9/S.E. of four animals

each.

N. Gupta et al. / Toxicology 188 (2003) 285�/296290

RR and MC-YR, three mice died. All the deathwas abrupt with sudden change in respiratory

variables. The mice that did not die during the 4-

h monitoring period did not show any change in

any of the variables, and they also survived.

Representative graph (one animal each for each

toxin) of the respiratory variables measured con-

tinuously after administration of the toxins is

given in Fig. 5.

4. Discussion

Many cyanobacterial species are capable of

producing potent toxins, which have been known

to cause intoxications and fatalities in wildlife,

livestock and humans. Microcystis is among the

most widely distributed genus and are known toproduce cyclic peptide toxins, the microcystins

(Carmichael, 1992; Hitzfeld et al., 2000). These

toxins are retained within the cyanobacterial cells

and are released only upon lysis of the cells during

the decomposition of cyanobacterial blooms. De-

tailed studies have not been carried out on toxic

manifestation of different microcystin variants. In

this study we investigated the comparative effectsof three most commonly and widely distributed

microcystin variants namely LR, RR and YR on

certain biochemical, respiratory and histological

variables in mice.

The acute LD50 determination of the three

variants showed variation in toxicity with MC-

LR being the most toxic followed by MC-YR and

MC-RR. There was significant difference betweentime and death of MC-YR compared with LR and

RR. The potent toxicity of the microcystins is

primarily due to marked inhibition of protein

phosphatase 1 and 2A (Eriksson et al., 1990).

Acute microcystin poisoning in mammals is char-

acterised by disruption of hepatic architecture

leading to massive intra hepatic haemorrhage

and death. In the present study all the three-microcystin variants caused significant increase in

LBI due to pooling of blood in the liver. Increase

in liver weight is a characteristic toxic effect of

microcystins and reported in a number of animal

models. Serum enzymes of hepatic origin have

shown significant increase in all the three toxins.

Serum levels of AST, ALT and g-GT showedsignificant increase compared with control as early

as 30 min post-exposure in all three toxins and was

further enhanced at MTD. Similarly increased

LDH levels were noticed at 30-min post treatment.

Increased serum activity of enzymes considered

indicative of hepatocellular damage has been

previously observed in both acute and chronic

toxicity studies (Rao et al., 1995; Nishiwaki-Matsushima et al., 1992). Increased serum ALT

activity is an important clinical indicator of

hepatocellular damage. It is a cytosolic enzyme

that is released into the blood following hepato-

cellular damage or necrosis. The results of our

study show that all three microcystin variants

induced time dependent increase in serum ALT

levels. The sensitivity of serum ALT activity as anindicator of hepatic damage may be decreased

secondary to decreased hepatic levels (Guzman

and Solter, 1999). It was shown that sub chronic

MC-LR exposure resulted in decreased hepatic

ALT protein and mRNA concentrations indicat-

ing one of the important toxic manifestation of

MC-LR could be decreased hepatic ALT synthesis

(Solter et al., 2000). The effects of down regulationof hepatic enzyme synthesis by microcystins on

hepatic function are not well known, however; it is

possible that the metabolic abilities of the liver

could be compromised.

The three toxins also caused depletion of hepatic

GSH levels with MC-LR causing significant de-

pletion compared with MC-RR and MC-YR. The

hepatic GSH content is critical factor for preser-ving normal cellular redox balance and protecting

the hepatocytes against oxidative stress. Cellular

reduced glutathione is important for the regulation

of cytoskeletal organisation. At present there are

several lines of evidence suggesting that GSH may

play a critical role in the detoxification of micro-

cystins. Microcystins could conjugate with GSH in

cell-free systems or in the liver tissue in vivo, whichsubstantially reduced its cytotoxicity (Ding et al.,

2000a). In the present study DNA fragmentation

was observed at 30 min post treatment in all the

three-microcystin variants. Significant further in-

crease was observed at MTD. The finding indi-

cates that DNA damage is an early event occurring

before necrotic liver damage. Microcystins in-

N. Gupta et al. / Toxicology 188 (2003) 285�/296 291

Fig. 4.

N. Gupta et al. / Toxicology 188 (2003) 285�/296292

duced DNA damage in mouse liver in vivo (Rao

and Bhattacharya, 1996) and in vitro in cultured

rat hepatocytes (Ding et al., 2000b), baby hamster

kidney cells and mouse embryo fibroblasts (Rao et

al., 1998), and Hep G2 cells (Bojana et al., 2003)

support the present results. The DNA damage

observed in the present study might be a conse-

quence of endonucleolytic DNA degradation as-

sociated with cytotoxicity, rather than by a direct

toxin�/DNA interaction.The morphological evidence of hepatocellular

injury by MC-LR, RR and YR was characterised

by apoptosis, centrilobular fibrosis, inflammation

and fatty vacuolar change. The induction of free

radical formation and mitochondrial alteration are

two major events found in microcystin treated

hepatocytes (Ding et al., 2000b). Immunohisto-

chemical localisation studies have shown that

positive immunostaining with anti-MC-MAb in

centrilobular hepatocytes in association with the

occurrence of haemorrhage or apoptosis. As

apoptotic hepatocytes would be finally phagocy-

tized and degraded by neighbouring hepatocytes

and macrophages, apoptotic cells/bodies contain-

ing MC-LR may have a role in elimination of the

toxin from the liver (Yoshida et al., 1998).

In the present study, in addition to histological

changes in the liver, all three toxins also showed

significant histological changes in lung like pul-

monary inflammation, congestion and haemor-

rhage. It is a normal practice to monitor

respiratory variables induced by a toxicant after

inhalation exposure. However, considering the

changes observed in lung histology we evaluated

the respiratory variables after i.p. administration

of toxin. Continuous monitoring of respiratory

variables after i.p. or p.o. administration can

reveal pulmonary effects (Sykes et al., 1977). All

three microcystin variants caused abrupt death

without much change in the respiratory variables

Table 3

Hepatic lesions caused by microcystin variants after i.p. administration of one LD50 dose in mice

Lesions MC-LR MC-RR MC-YR

30 min MTD 30 min MTD 30 min MTD

Basophilic stipling �/ �/�/ �/ �/�/ �/�/ �/�/�/

Degeneration and vacuolation of hepatic parenchyma �/�/ �/�/�/ �/ �/�/ �/�/ �/�/�/�/

Portal mononuclear cell infiltration �/ �/�/ �/ �/�/ �/�/�/ �/�/�/�/

Congestion and haemorrhage �/�/ �/�/�/ �/�/ �/�/�/ �/�/�/ �/�/�/�/

Hepatic vacuolation �/ �/�/ �/ �/�/ �/�/ �/�/�/

Granulatory cytoplasm �/ �/�/�/ �/ �/ �/�/ �/�/�/

Eosinophilic debris �/ �/�/ �/ �/ �/�/�/ �/�/�/�/

Hyper activation of Kupffer cells �/�/ �/�/�/ �/ �/�/ �/�/�/ �/�/�/�/

Accumulation of hemosiderin �/�/ �/�/�/ �/ �/�/ �/�/�/ �/�/�/�/

Scoring: �/, None, �/, Minimal (B/12%), �/�/, Mild (B/22%), �/�/�/, Moderate (B/45%), �/�/�/�/, Severe (�/45%). MTD, mean

time to death.

Fig. 4. Liver (A�/D) and lung (E�/H) histology of microcystin variants treated mice. Tissue samples were taken at time point before

death. H&E; �/100. (A) Control liver showing normal hepatic cord pattern of lobules, hepatocytes, central canal and bile duct; (B)

liver from MC-RR treated mice showing mild congestion, haemorrhage, degeneration and vacuolation of hepatic parenchyma; (C)

liver from MC-YR treated mice showing severe vacuolative degeneration, congestion, haemorrhage and accumulation of hemosiderin

(arrow); (D) liver from MC-LR treated mice showing degenerative changes with congestion, haemorrhage (arrow) and granulation of

cytoplasm. (E) Control mice lung showing homogenous parenchyma with normal airways, air sacs, alveolar septa and bronchiole; (F)

lung from MC-RR treated mice showing focal degeneration and sloughing off bronchiole parenchyma (arrow); (G) lung from MC-YR

treated mice showing severe bronchial parenchyma cell degeneration and sloughing off cells leading to occlusion of bronchiole (arrow);

(H) MC-LR treated mice showing inflammatory reaction and accumulation of eosinophilic edematous fluid.

N. Gupta et al. / Toxicology 188 (2003) 285�/296 293

during the observation period, showing that they

do not have any direct effect on the respiratory

system. The chemicals that cause direct effect on

the pulmonary epithelium may induce sensory or

pulmonary irritation, and those that alter the

sympathetic or the parasympathetic nervous sys-

tem may show broncho dilatation or broncho

constriction (Vijayaraghvan et al., 1994). Slatkin

et al. (1983) proposed that atypical pulmonary

thrombi might account for pulmonary congestion

and heart failure, resulting in centrilobular hepatic

necrosis and haemorrhage. Mild pulmonary

edema, peritoneal fluid accumulation, enteritis

with haemorrhage have been reported in response

to microcystin exposure in a range of species. The

lungs of intra vascularly dosed MC-LR in swine

remained effective in gas exchange, as reflected by

high pO2 and low pCO2 values (Beasley et al.,

2000).

Modifications at different sites of the micro-

cystin molecule were found to produce mild to

marked changes in toxicity. Harada et al. (1990)

described geometrical isomers of microcystin-LR

and RR, which were nonlethal when administered

i.p. to mice at 1.2 mg/kg. Replacement of second

amino acid leucine in MC-LR with tyrosine (MC-

YR) or arginine (MC-RR) still retained hepato-

toxicity. When L-leucine, which is a hydrophobic

amino acid is replaced by other hydrophobic

amino acids, i.e. Phe, Ala or Trp the resulting

microcystin retained LD50 from 171 to 249 mg/kg

(Stotis et al., 2000). The differences between

hydrophilic and hydrophobic microcystins could

result in changes in organotropism, toxicokinetics

and bioaccumulation (Pia and Meriluoto, 2003).

Apart from the protein phosphatase inhibitory

activity of microcystins some reports are available

on other toxic manifestation of microcystin var-

iants. The infusion of macrophages supernatants

stimulated by microcystin-LR caused significant

increase in renal vascular resistance, glomerular

filtration rates and urinary flow (Nobre et al.,

2003). Lankoff and Kolataj (2000) showed inhibi-

tory action of MC-YR on certain proteases that

affects the stability of lysosomal membranes and

provoke autodestruction of the cell and severe

damage of the liver. Mikhailov et al. (2003)

recently identified ATP-synthase as a novel in-

tracellular target for microcystin-LR and observed

that most of the physiological effects of micro-

cystin exposure are still only secondary to protein

phosphatase inhibition. In the present study

though there are minor variations in some of the

biochemical variables and histological effects, the

pronounced hepatotoxicity was observed in all the

three toxins. In conclusion, results of our study

indicate in addition to potent toxicity of micro-

cystin-LR, other two common variants MC-YR

Table 4

Pulmonary lesions caused by microcystin variants after i.p. administration of one LD50 dose in mice

Lesions MC-LR MC-RR MC-YR

30 min MTD 30 min MTD 30 min MTD

Bronchial epithelial hyperplasia �/ �/�/ �/ �/�/ �/�/�/ �/�/�/�/

Bronchial epithelial necrosis �/�/ �/�/�/ �/�/ �/�/�/ �/�/�/ �/�/�/�/

Occlusion bronchiole �/�/ �/�/�/ �/ �/�/ �/�/ �/�/�/

Haemorrhage �/ �/�/ �/ �/�/ �/�/ �/�/�/�/

Congestion �/�/ �/�/ �/ �/�/ �/�/ �/�/�/

Stinosis of bronchial lumen �/ �/�/ �/ �/�/ �/�/ �/�/

BALT necrosis �/ �/�/ �/ �/ �/�/ �/�/�/

Inflammatory reaction �/ �/�/ �/ �/�/ �/�/ �/�/�/

Edema �/ �/ �/ �/ �/ �/�/

Accumulation of fibrinoid debris �/ �/�/ �/ �/ �/�/ �/�/�/

Scoring: �/, None, �/, Minimal (B/12%), �/�/, Mild (B/22%), �/�/�/, Moderate (B/45%), �/�/�/�/, Severe (�/45%); MTD, mean

time to death.

N. Gupta et al. / Toxicology 188 (2003) 285�/296294

and MC-RR are also highly toxic and could pose

significant environmental health hazard to live-

stock and humans when present in potable waters.

Acknowledgements

The authors thank Mr. K. Sekhar, Director,

DRDE, for his support and keen interest. We

thank Professor W.W. Carmichael, Department of

Aquatic Biology, Wright State University, OH,

USA for providing Microcystins. Ms. Nidhi Gup-

ta is thankful to Defence Research and Develop-ment Organisation (DRDO), for the award of

Research Fellowship.

References

Amorin, A., Vasconcelos, V., 1999. Dynamics of microcystin in

the mussel Mytilus galloprovincialis . Toxicon 37, 1041�/

1052.

Azevedo, S.M.F.O., Carmichael, W.W., Jochimsen, E.M.,

Rinehart, K.L., Lau, S., Shaw, G.R., Eaglesham, G.K.,

2002. Human intoxication by microcystins during renal

dialysis treatment in Caruaru, Brazil. Toxicology 181�/182,

441�/446.

Beasley, V.R., Lovell, R.A., Holmes, K.R., Walcott, H.E.,

Schaeffer, D.J., Hoffmann, W.E., Carmichael, W.W., 2000.

Microcystin-LR decreases hepatic renal perfusion, and

causes circulatory shock, severe hypoglycaemia, and term-

inal hyperkalemia in intravascularly dosed swine. J. Toxicol.

Environ. Health 61, 281�/303.

Bell, S.G., Codd, G.A., 1994. Cyanobacterial toxins and human

health. Rev. Med. Microbiol. 5, 256�/264.

Bojana, Z., Bojan, S., Metka, F., 2003. Microcystin-LR induces

oxidative DNA damage in human hepatoma cell line

HepG2. Toxicon 41, 41�/48.

Carmichael, W.W., 1992. Cyanobacteria secondary metabo-

lites-cyanotoxins. J. Appl. Bacteriol. 72, 445�/459.

Chorus, I., Falconer, I.R., Salas, H.J., Batram, J., 2000. Health

risks caused by freshwater cyanobacteria in recreational

waters. J. Toxicol. Environ. Health 3, 323�/347.

Codd, G.A., Bell, S.G., Brooks, W.P., 1989. Cyanobacterial

toxins in water. Water Sci. Technol. 21, 1�/13.

Dawson, R.M., 1998. Toxicology of microcystins. Toxicon 7,

953�/962.

Ding, W.X., Shen, H.M., Ong, C.N., 2000a. Microcystic

cyanobacteria extract induces cytoskeleton disruption and

intracellular glutathione alteration in hepatocytes. Environ.

Health Perspect. 108, 605�/609.

Ding, W.X., Shen, H.M., Ong, C.N., 2000b. Critical role of

reactive oxygen species and mitochondrial permeability

transition in microcystin-induced rapid apoptosis in rat

hepatocytes. Hepatology 32, 547�/555.

Dixon, W.J., 1965. The up and down method for small samples.

J. Am. Stat. Assoc. 60, 967�/978.

Eriksson, J.E., Toivola, D., Meriluoto, J.A.O., Karaki, H.,

Han, Y., Hartshorne, D., 1990. Hepatocyte deformation

induced by cyanobacterial toxin reflects inhibition of

protein phosphatases. Biochem. Biophys. Res. Commun.

173, 1347�/1353.

Guzman, R.E., Solter, P.F., 1999. Hepatic oxidative stress

following prolonged sub lethal microcystin-LR exposure.

Toxicol. Pathol. 27, 582�/588.

Harada, K.I., Ogawa, K., Matsuura, K., Hideaki, M., Suzuki,

M., Watanabe, M.F., Itezono, Y., Nakayama, N., 1990.

Structural determination of geometrical isomers of micro-

Fig. 5. Representative graph (one animal each for each

microcystin) of percent change of respiratory variables after

i.p. administration of one LD50 (arrow). VT, tidal volume; Ti,

time of inspiration; TE, time of expiration and f , respiratory

frequency. The control period values are VT�/0.0849/0.005

(ml); TI�/0.1159/0.003 (s); TE�/0.1639/0.003 (s) and f�/

2319/5.

N. Gupta et al. / Toxicology 188 (2003) 285�/296 295

cystin LR and RR from cyanobacteria by two-dimensional

NMR spectroscopic techniques. Chem. Res. Toxicol. 3,

473�/481.

Hisin, P.J., Hilf, R., 1976. A fluorometric method for determi-

nation of oxidised and reduced glutathione in tissues. Anal.

Biochem. 74, 214�/226.

Hitzfeld, B.C., Hoger, S.J., Dietrich, R., 2000. Cyanobacterial

toxins: removal during drinking water treatment, and

human risk assessment. Environ. Health Perspect. 10,

113�/122.

Lankoff, A., Kolataj, A., 2000. Influence of microcystin-YR

and nodularin on the activity of some proteolytic enzymes

in mouse liver. Toxicon 39, 419�/423.

Lowry, O.H., Rosenberg, N.J., Farr, A.L., Randall, R.J., 1951.

Protein measurement with the Folin phenol reagent. J. Biol.

Chem. 193, 265�/275.

Mikhailov, A., Harmala-Brasken, A., Hellman, J., Meriluoto,

J., Eriksson, J.E., 2003. Identification of ATP-synthase as a

novel intracellular target for microcystin-LR. Chem.-Biol.

Interact. 142, 223�/237.

Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S., Suga-

numa, M., Kohyama, K., Ishiwaka, T., Carmichael,

W.W., Fujiki, H., 1992. Liver tumour promotion by the

cyanobacterial cyclic peptide toxin microcystin-LR. J.

Cancer Res. Clin. Oncol. 118, 420�/424.

Nobre, A.C.L., Martins, A.M.C., Havt, A., Benevides, C.,

Lima, A.A.M., Fonteles, M.C., Monteiro, H.S.A., 2003.

Renal effects of supernatant from rat peritoneal macro-

phages activated by microcystin-LR: role protein mediators.

Toxicon 41, 377�/381.

Pia, S.M., Meriluoto, V., 2003. Interaction between microcys-

tins of different hydrophobicities and lipid monolayers.

Toxicon 41, 349�/355.

Rao, P.V.L., Bhattacharya, R., 1996. The cyanobacterial toxin

microcystin-LR induced DNA damage in mouse liver in

vivo. Toxicology 114, 29�/36.

Rao, P.V.L., Bhattacharya, R., Pant, S.C., Bhaskar, A.S.B.,

1995. Toxicity evaluation of in vitro cultures of freshwater

cyanobacterium Microcystis aeruginosa : I Hepatic and

histopathological effects in rats. Biomed. Environ. Sci. 8,

254�/264.

Rao, P.V.L., Bhattacharya, R., Parida, M.M., Jana, A.M.,

Bhaskar, A.S.B., 1998. Freshwater cyanobacterium Micro-

cystis aeruginosa (UTEX 2385) induced DNA damage in

vivo and in vitro. Environ. Toxicol. Pharmacol. 5, 1�/6.

Rao, P.V.L., Bhaskar, A.S.B., Vijayaraghavan, R., 1999.

Sulphur mustard induced DNA damage in mice after

dermal and inhalation exposure. Toxicology 139, 39�/51.

Slatkin, D.N., Stoner, R.D., Adams, W.H., Kycia, J.H.,

Siegelman, H.W., 1983. Atypical pulmonary thrombosis

caused by a toxic cyanobacterial peptide. Science 220,

1383�/1385.

Solter, P.F., Zonglin, L., Guzman, R., 2000. Decreased hepatic

ALT synthesis is an outcome of sub-chronic microcystin-LR

toxicity. Toxicol. Appl. Pharmacol. 164, 216�/220.

Stotis, R.S., Namikoshi, M., Haschek, W.M., Rinehart, K.L.,

Carmichael, W.W., Dahlem, A.M., Beasley, V.R., 2000.

Structural modifications imparting reduced toxicity in

microcystins from Microcystis spp. Toxicon 31, 783�/789.

Sykes, B.I., Purchase, I.F., Smith, L.L., 1977. Pulmonary

ultrastructure after oral and intravenous dosage of paraquat

to rats. J. Pathol. 121, 233�/241.

Vijayaraghavan, R., Schaper, M., Thompson, R., Stock, M.F.,

Boylstein, L.A., Luo, J.E., Alarie, Y., 1994. Computer

assisted recognition and quantification of the effects of

airborne chemicals acting at different areas of respiratory

tract in mice. Arch. Toxicol. 68, 490�/499.

Wyllie, A.H., 1980. Glucocorticoid induced thymocyte apop-

tosis is associated with endogenous endonuclease activation.

Nature 284, 555�/556.

Yoshida, T., Makita, Y., Tsutsumi, T., Nagata, S., Tashiro, F.,

Yoshida, F., Sekijima, M., Tamura, S.I., Harada, T., Maita,

K., Ueno, Y., 1998. Immunohistochemical localization of

microcystin-LR in the liver of mice: a study on the

pathogenesis of microcystin-LR induced hepatotoxicity.

Environ. Toxicol. Pathol. 26, 411�/418.

Yu, S.Z., 1995. Primary prevention of hepatocellular carci-

noma. J. Gastroenterol. Hepatol. 10, 674�/682.

N. Gupta et al. / Toxicology 188 (2003) 285�/296296