Radiol Oncol 2002; 36(2): 165-7. 

Chronic exposure to cyanobacterial lyophilisate reveals stronger 
effects than exposure to purified microcystins – a MRI study 

Dušan Šuput1, Aleksandra Milutinovič1, Igor Serša2 and Bojan Sedmak3 

1School of Medicine, Institute of Pathophysiology, 1001 Ljubljana, Slovenia; 2Inštitut Jožef Stefan, 
Jamova 39, 1000 Ljubljana, Slovenija; 3Department of Ecotoxinology and Ecotoxicology, 
National Institute of Biology, Večna pot, 1000 Ljubljana, Slovenia 


Introduction 

Microcystins are potent hepatotoxins, tumor 
promoters, and carcinogens. Although they 
are present in surface water bodies worldwide 
little has been done to assess the effects of 
chronic exposure to these substances in human 
population. This may partly be due to 
the fact that acute intoxication by these substances 
is rare in man. Recent experience 
from Brazil where a number of dialysis patients 
died due to the presence of microcystins 
in the water used for dialysis is a serious warning. 
Chronic exposure to microcystins is less 
dramatic, but more widespread. Therefore 
the aim of our study was to test magnetic resonance 
imaging (MRI) as a noninvasive and 
harmless method for the early detection of 
changes in liver tissue after chronic exposure 
to microcystins. It should be emphasized that 
living organism are in most cases chronically 
exposed to toxic cyanobacteria and/or to their 
dissolved contents, which consist of a broad 
range of biologically active substances and 
not only microcystins.1-4 Therefore the second 
aim of our study was to assess the possible 
synergistic interactions between these 
substances in chronic intoxication by using 
either purified microcystins or cyanobacterial 
lyophilisate (CL). 

Methods 

Male albino rats (Wistar) weighing 165-190 g 
were used in all experiments. The animals 
were kept at standard room condition (room 
temperature 24 °C, daily-night interval at 12 
hours, exposed to 60 lux artificial light). The 
number of animals was kept at minimum. 
The experiments were performed with the guidelines 
of IST. Either cyanobacterial lyophilisate 
or purified microcystins were injected 

i.p. in the intervals of 3 days for 2 months, the 
total cumulative dose of microcystins (either 
in the lyophilisate or purified substance) was 
2 LD50. 
Magnetic resonance imaging of the rat’s liver 
was performed on a Bruker Biospec system 
with a 2.35 T horizontal bore magnet on 
animals anesthetized with i.p. injection of 
Xylazin (15 mg/kg), Ketanest (100 mg/kg) and 
atropine (0.3 mg/kg). In this study, standard 
T1 weighted spin-echo magnetic resonance 
imaging method with echo time 18 ms and repetition 
rate 400 ms was used. Signal averaging 
of 10 signal acquisitions was used to improve 
the signal to noise ratio. In order to 
extract the liver volume, rats were imaged in 
seven consecutive slices in two experiments, 
first in transverse and then in coronal slice 
orientation. Imaging filed of view and slice 
thickness was at each experiment adjusted so 
that imaging volume covered the whole liver 
region. Thus, field of view was in the range 810 
cm and slice thickens 3-4 mm. T1 weig



Šuput D et al. / Chronic exposure to cyanobacterial lyophilisate 

hted MR images had good contrast between 
liver and surrounding tissues that allowed 
precise liver region selection and its area calculation 
for each slice. Liver volume was later 
calculated as a sum of liver areas in all slices 
multiplied by the slice thickness. 

Results and discussion 

In acute experiments MRI revealed enlargement 
of liver in both groups of experimental 
animals, and there was no difference whether 
they received only purified microcystins or 
the same amount of microcystins present in 
the CL. Significant differences have been observed 
between the group that received CL or 
purified microcystins only in the chronic experiments. 
Liver from the animals injected 
with the microcystin- LR showed only minor 
changes on the signal intensity on MRI images, 
and patho -morphological examination of 
abdominal organs on sacrificed animals showed 
degenerative changes in one animal only. 
The MR images of the liver from all animals 
injected with CL showed irregular 
changes in the signal intensity and nodular 
formations have been observed. Patho -morphological 
examination of sacrificed animals 
showed granular structure of liver edges, and 
enlargement of kidneys. However, degenerative 
changes characterised as periportal fibro


sis and fatty infiltrations were observed in 
both groups of chronically treated animals. 
Changes were more pronounced in the case 
of the animals treated with CL. No tumours 
were detected in any of the treated animals, 
which is in agreement with the data from other 
authors5 that showed that longer exposures 
and higher dosages are necessary to produce 
carcinogenic effects. 

Extracts from toxic cyanobacteria induce 
DNA damage in vitro as well as in vivo.7 This 
implies that microcystins can no longer be 
treated merely as tumour promoters. The enlargement 
of kidney shown in our experiments 
suggests that exposure to toxic cyanobacteria 
also affects other organs, which has 
already been proposed.8 Microcystins could 
be responsible for these effects, but the combined 
action of the constituents of cyanobacteria 
is evidently important as kidney enlargement 
has been observed only in the group of 
animals treated with CL. The data show that 
not only microcystins but also other components 
of the cyanobacterial bloom can affect 
the health of population.6 Therefore the health 
risk of hepatotoxic cyanobacteria estimated 
from the microcystin content is underestimated. 



Figure 1. MR images of the liver exposed to cyanobacterial lyophilisate or to purified microcystins alone. A. Control 
liver is of a dark gray and homogeneous colour. B: Chronic exposure to cyanobacterial lyophilisate (CL) results 
in liver degeneration shown as non-homogeneous signal from the liver seen as white patches. C) Chronic exposure 
to microcystins gives rise to only slight changes in the liver structure as seen by MRI. 

Radiol Oncol 2002; 36(2): 165-7. 


Šuput D et al. / Chronic exposure to cyanobacterial lyophilisate 

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