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Cyanobacterial extracts and microcystin-LR are inactive in the micronucleus assay in vivo and in vitro
Mutation Research 699 (2010) 5–10
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Cyanobacterial extracts and microcystin-LR are inactive in the micronucleus assay in vivo and in vitro Lilianne Abramsson-Zetterberg a,∗ , Ulla Beckman Sundh a , Roland Mattsson b a b
The Swedish Food Administration, Box 622, 751 26 Uppsala, Sweden Department of Pathology and Wildlife, National Veterinary Institute, 751 89 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 2 November 2009 Received in revised form 22 January 2010 Accepted 29 January 2010 Available online 8 April 2010 Keywords: Microcystin-LR Cyanobacteria extract Micronuclei Flow cytometer
a b s t r a c t Cyanobacteria are sometimes widespread in lakes and can produce potent toxins, which can be dangerous for animals that drink the water, e.g. cattle and dogs. If the toxins are taken up by fish and other organisms in the food chain, or occur in drinking-water, they may pose a problem also for humans. Microcystin-LR, a hepatotoxic cyclic peptide, is one of the most frequently found cyanobacterial toxins. Data on the genotoxic potential of microcystin-LR and other cyanobacterial toxins are contradictory. Here we report results of the micronucleus assay carried out in vivo and in vitro with these toxins. To increase the sensitivity, we used the flow cytometry-based micronucleus assay in the mouse. In this study both pure microcystin-LR and cyanobacterial extracts originating from four different lakes in Sweden were analysed. Although doses up to near lethality were used and an average of 200,000 young erythrocytes, polychromatic erythrocytes, were analysed from each animal, no genotoxic effect was observed, nor could any effect be shown in the in vitro micronucleus study, using human lymphocytes. These results show that the low concentration of microcystins that now and then occur in drinkingwater does not increase the cancer risk through chromosome breaks or mal-distribution of chromosomes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cyanobacteria are sometimes widespread in lakes and can produce potent toxins. Several species of cyanobacteria produce different kinds of toxins. It is estimated that a large part of all water blooms are toxic. The production of cyanobacteria will increase in a warmer climate and when more nutrients leak into lakes from agricultural manuring. Animals, e.g., cattle and dogs that drink the water may be intoxicated. If the toxins are taken up by fish and other sea-food they may pose a problem also for humans. Contamination with cyanobacteria may also be a problem in drinking-water obtained from surface waters. Many bacterial toxins are eliminated in the refining process in the water plants. In spite of this, measurable amounts of microcystins may remain in the water. The exposure will be even higher for people who use this water for drinking without any filtering. The neurotoxic and/or hepatotoxic effects of the common toxins produced by some species of cyanobacteria are well known. The toxic effects are likely to have a threshold concentration below which no effect occurs. Thus the WHO suggests a tolerable daily intake (TDI) for microcystin-LR of 0.04 g/kg bw/day, a value
∗ Corresponding author. Tel.: +46 18 17 57 63; fax: +46 18 17 14 33. E-mail address: [email protected] (L. Abramsson-Zetterberg). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.04.001
assumed to safeguard against the neurotoxic and/or hepatotoxic effects [1]. However, if cyanobacterial toxins would be carcinogenic due to a genotoxic mechanism, even lower concentrations would be a potential problem. Convincing results about a carcinogenic effect of these substances have not been published so far. However, it is often stated in the literature that microcystins have tumour promoting and possibly also genotoxic or carcinogenic properties. In an early study, Falconer and co-worker [2,3] report indications of tumour-promoting activity when an extract of a naturally occurring toxic Microcystis bloom was given orally to mice initiated topically with dimethylbenzanthracene. There was an increase of the weight of skin tumours in these mice. The composition of the extract was, however, not defined and since microcystins are mainly taken up in the liver it would be surprising if these substances act as effective skin-tumour promoters. In a study by Nishiwaki-Matsushima et al. [4] the authors report tumourpromoting activity of microcystin-LR in livers of rats that had been initiated with diethylnitrosamine and partially hepatectomized. Increasing intraperitoneal doses of microcystin-LR corresponded with increasing numbers and areas of glutathione S-transferase (placental form)-positive foci in the livers, which was interpreted as an indication of tumour-promoting activity. Although this study is often cited as proof of the tumour-promoting activity of microcystins, it must be noted that glutathione S-transferase-positive foci are not an expression of tumour promotion per se. Other stud-
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ies have failed to show tumour-promoting or -initiating activity of microcystin-LR or of extracts of Microcystis, when applied orally [5–7]. However, when microcystin-LR was given via intraperitoneal injection during 28 weeks, the mice developed liver nodules [8]. Since there is uncertainty about the mechanism behind the interaction between microcystin-LR and the cells exposed, WHO has suggested an uncertainty factor of 1000 and a provisional guideline value of 1 g/l [1]. Published data about the genotoxic potential of microcystinLR and other cyanobacterial toxins are contradictory. When it can be clearly demonstrated that microcystin-LR is not mutagenic, the uncertainty factor may be lower. In order to contribute to the database concerning the suggested guideline concentration for microcystin-LR in drinking-water, we report results from some short-term tests using the micronucleus assay in vivo and in vitro. Both bacterial extracts and pure microcystin-LR were studied. To increase the sensitivity, we used the flow cytometry-based micronucleus assay in the mouse. 2. Material and methods This study comprises a total of three different experiments: two in vivo and one in vitro micronucleus study. In the first in vivo study (Experiment 1) the mice were exposed to different doses of microcystin-LR; in the second in vivo study (Experiment 2) the mice were exposed to extracts from four different samples of cyanobacteria (blue-green algae); in the third experiment (Experiment 3) the four cyanobacterial extracts were analysed in the in vitro micronucleus study with human lymphocytes. The extract samples originated from four lakes in Sweden, samples 1 and 3 from the southern and the northern part of the lake Mälaren (in the vicinity of Stockholm), sample 2 from the very southern part of Sweden, Skåne, and sample 4 from a lake in Småland (southern Sweden).
2.1. Collection, identification, and preparation of cyanobacterial samples The cyanobacteria were collected in the middle of the summer of 1985 (samples 1, 2, and 3) and 1987 (sample 4) from four different inland waters during the water-bloom period. Microscopic examination revealed that the dominating species in the respective samples were as follows: sample 1: 99% Planktothrix agardhii; sample 2: 80% Microcystis viridis, 20% Microcystis wesenbergi; sample 3: 99% Microcystis aeruginosa; sample 4: 85% Anabaena flos-aquae, 15% M. aeruginosa + Pl. agardhii + Gomphosphaeria sp. All samples were freeze-dried directly after examination and stored at −20 ◦ C. The toxins are stable and freeze-dried samples have been retested over time. For the toxicity test (bioassay) 250 mg of the freeze-dried sample were mixed in 5.0 ml NaCl (0.9%) for 15 min before being centrifuged at 10 min at 3200 × g. One milliliter of the supernatant was injected intraperitoneally in mice for the toxicity test. The mice were kept under observation and the symptoms, time of death, and the subsequent autopsy indicated the occurrence of toxins. Also a 10-fold lower dose, 0.1 ml of the toxin, resulted in an acute toxic effect. Prior to the genotoxicity tests, freeze-dried samples were extracted in the same way as for the tests of acute toxicity. The mice were given about half the lowest dose that resulted in death. The occurrence of different microcystins (Mic-LR, -YR, -RR) in cyanobacterial bloom samples was determined by high-performance liquid chromatography (HPLC) with diode-array detection. The analytical method used was based on a standard technique and an in-house method developed for freeze-dried algal samples [9]. HPLC analyses were performed on an Ace3 C18 reversed-phase 10-cm × 4.6-mm column with a Shimadzu LC10AD Vp chromatograph and Shimadzu diode-array detector SPD M10A Vp. Identification of the microcystin peaks was based on a comparison of retention times and absorbance spectra for samples and standard solutions of microcystins. Also a reference material of freeze-dried algae was analysed together with the samples. The concentrations of microcystins in the samples analysed were in the range 2–340 ng/mg.
2.2.2. Chemicals Microcystin-LR (CAS no. 101043-37-2), colchicine (CAS no. 64-86-8), and Hoechst 33342 (HO342) were purchased from Sigma–Aldrich, Sweden; Percoll was from Amersham Biosciences, Sweden; Fluothane from Astra, Sweden; and Thiazole orange (TO) from Molecular Probes, OR, USA. 2.2.3. Experimental design and sampling The genotoxic effects in vivo of microcystin-LR and of the four cyanobacterial extracts were determined using the flow cytometry-based micronucleus assay in mice. In both in vivo studies the animals were randomly divided in different groups. In all experiments the mice were injected intraperitoneally (i.p.) with a single dose of 10 l/g bw. Microcystin-LR and the cyanobacterial extracts were diluted in PBS just prior to injection. The positive control mice received injections of 1 mg/kg bw colchicine dissolved in PBS. In both in vivo studies (Experiments 1a and 1b) the mice were anaesthesized with Fluothane and blood samples were drawn from the orbital vein into heparinized tubes 42 h after injection. Directly after blood sampling the animals were killed by cervical dislocation. The sampling time was based on the known time interval between appearance of polychromatic erythrocytes (PCE) in the bone marrow and in peripheral blood [10]. In Experiment 1, which involved i.p. administration of microcystin-LR to male CBA mice, a total of 26 mice were used. They were given the following doses of microcystin-LR: 0, 10, 20, 30, 34, 38, 42, 46, 50, and 55 g/kg bw. All groups consisted of two mice except those given a dose of 0 and the highest doses, 46, 50, and 55 g/kg bw, and the positive control (colchicine), which all comprised three mice. Two out of the 26 mice died during the experiment. These mice had received the highest doses. Experiment 2, with i.p. administration of cyanobacterial extract to male CBA mice, involved 28 mice. For each of the four extracts, five mice were used. All five mice given the same extract received the same high dose. Results of the bioassay (see Section 2.1) where the acute toxicity of these four cyanobacterial extracts were tested, showed that a dose, two times used in this study, of 5 mg lyophilized algal cells/mouse was lethal. Taking these results into consideration we chose a dose of 2.0 mg of lyophilized algal cells/mouse. Three mice in the positive control group were given colchicine, and five mice constituted the negative control group and received PBS. 2.2.4. Purification, fixation and staining of erythrocytes The methods for purification, fixation and staining of erythrocytes have been published previously [11,12]. Briefly, 5 l of whole blood was layered on a 65% Percoll gradient and centrifuged. The supernatant was carefully removed and the pellet was re-suspended in PBS and fixed in glutaraldehyde according to a method described by Hayashi et al. [13]. The samples were coded and stored at 4 ◦ C for a few days. A staining solution, containing the fluorescent dyes HO342 (DNA-dye) and TO (RNA-dye) in PBS, was added to the fixed cells. The staining was performed 1 day prior to analysis. 2.2.5. Flow-cytometric analysis and the determination of micronucleus frequency For the analysis a FACSVantage SE flow cytometer (Becton-Dickinson Immunocytometry Systems, Sunnyvale, CA) was used according to a method described elsewhere [11,12]. The cells were automatically analysed when they, one by one, passed through two laser beams (350 and 488 nm). Information about size and structure was collected and used to exclude remaining nucleated cells as well as debris in the sample from further analysis. DNA- and RNA-content was detected as fluorescence from the two dyes HO342 and TO, respectively. RNA content, measured as the signal from the TO dye, was used to distinguish between young and old erythrocytes, PCE and NCE (poly- and normochromatic erythrocytes), respectively. The ratio of young to old cells gives an indication of cell proliferation (%PCE). This analysis was based on the information from about 20,000 cells per sample. Furthermore, since erythrocytes normally do not contain any DNA, a signal from the HO342 dye implies that it contains a micronucleus. Limiting the analysis to only PCE enabled us to calculate the frequency of micro-nucleated PCE. About 200,000 PCE were scored per animal. All analyses were performed using CellQuest software (Becton-Dickinson). Scatter plots of the information about DNA content vs RNA content were displayed for each analysed sample. Regions were defined for NCE, PCE, and MNPCE (micro-nucleated PCE), respectively. On the basis of this, the number of events in each region was determined. Frequencies of PCE and MNPCE were calculated. 2.3. Micronucleus assay in vitro in human lymphocytes (Experiment 3)
2.2. Micronucleus assay in vivo in mice (Experiments 1 and 2) 2.2.1. Animals Male CBA mice, 6–7 weeks old, weighing approximately 25 g, were obtained from Scanbur AB, Sollentuna, Sweden. The animals were housed at the Swedish National Food Administration in a 12 h light/12 h dark cycle with free access to solid food and tap water. All mice were acclimatised 1 week before treatment. The experiment was reviewed and approved by Uppsala Ethical Committee on Animal Experiments, application C295/6.
2.3.1. Chemicals F-10 HAM’s medium and fetal calf serum were from HyClone USA; penicillin/streptomycin (PEST) and l-glutamine from Bio Whittaker Europe; phytohaemagglutinin (PHA), cytochalasin B, and colchicine (CAS no. 64-86-8) from Sigma–Aldrich, Sweden. 2.3.2. Experimental design and treatment One experiment was performed with blood from a healthy non-smoking female, 55 years old. Whole blood cultures were treated with the four cyanobacterial
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Fig. 1. Results of the analysis by flow cytometry of polychromatic erythrocytes (PCE) from peripheral blood of male CBA mice given single injections (i.p.) with different amounts of microcystin-LR. Blood samples were taken 42 h after injection. Each data point is the mean value of fMNPCE for two mice, except for the mice given a dose of 0 and 46 g/kg bw, where each of the data points represents three mice. Two mice in each of the highest dose groups died during the experiment. There is no significant dose-response trend (see Table 1). The fMNPCE (frequency of micro-nucleated polychromatic erythrocytes in peripheral blood) for the mice given colchicine (1 mg/kg bw) was significantly different from the control level (p < 0.01).
extracts dissolved in PBS. Concentrations of 0, 0.25, 0.5, 1.0, and 2.0 mg extract of freeze-dried cyanobacteria per ml cell culture were used for each of the four extracts, respectively. Colchicine, final concentration 75 nM, was used as a positive control. The experiments were carried out according to the method described by Fenech and Morley [14] and refined by Migliore et al. [15]. Briefly, whole blood was cultured in F-10 HAM’s medium supplemented with 12% fetal calf serum, 0.5% l-glutamine, 2% PEST, and 1.2% PHA for 72 h. The cultures were kept in a humidified environment with 5% CO2 at 37 ◦ C. After 24 h of incubation, the different agents were added according to the experimental setup. After 44 h cytochalasin B was added to the cultures to a final concentration of 6 g/ml. The incubation continued until 72 h after initiation. The cultures were then centrifuged and the supernatant was removed. Erythrocytes were lysed by addition of KCl (0.075 M). After 3 min, prefixative (methanol/acetic acid 3:1, v/v) was added. The cells were rinsed and fixed in methanol and methanol/acetic acid (5:1, v/v) and spread on clean, cold, wetted glass slides. After drying, the cells were stained with 3% Giemsa dye. 2.3.3. Scoring All slides were randomised and coded prior to analysis. The scoring was performed in accordance with Fenech [16] using a Leica Ortholux II microscope. About 4000 bi-nucleated lymphocytes (cells that have undergone one mitotic division during the exposure) from each culture were scored for the presence of micronuclei. As a measure of cell division the proportion of bi-nucleated cells (% BinLymph) among the total of viable cells was calculated. About 600 cells per culture were scored for this purpose.
3. Results and discussion In this study the frequency of micro-nucleated cells was analysed in vivo and in vitro after exposure to both pure microcystin-LR and cyanobacterial extracts from four different lakes in Sweden. All four extracts showed acute toxicity in the mouse bioassay (see Section 2.1). None of the animals given different doses of microcystin-LR or the cyanobacterial extracts, showed an increased frequency of micro-nucleated polychromatic erythrocytes in the peripheral blood, fMNPCE (see Tables 1 and 2 and Fig. 1). Neither did the percentage of polychromatic erythrocytes, %PCE, reveal any changes in the cell proliferation (Tables 1 and 2). In the in vitro assay, an increased concentration of the extracts did not result in a concomitantly higher frequency of micro-nucleated bi-nucleated human lymphocytes, fBinMN (Table 3). In both the in vivo and in vitro studies sufficiently high doses and concentrations were analysed. At the highest doses of microcystin-LR or of the extract, an adverse effect was noticed, i.e., some mice died and some showed signs of distress (fur condition, wakefulness, etc.). In addition, the concentrations used in the in vitro study were limited because it was not possible to dissolve higher amounts of the extracts. Only the
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mice and cells exposed to the positive control, colchicine, showed a significant increase in the mean fMNPCE or fBinMN. The HPLC analysis showed that three of the four extracts contained different kinds of microcystin. Three microcystins (Mic) could be quantified, i.e., Mic-RR, -LR, and -YR. The analysis showed that sample 1 contained about 70% Mic-LR, 30% Mic-YR, <1% MicRR; sample 2: 54% Mic-RR, 40% Mic-LR, 6% Mic-YR; sample 3: 40% Mic-RR, 60% Mic-LR, <1% Mic-YR. Sample 4 mainly contained toxins other than microcystins. According to the algal analysis and from observation of the mice, the major toxin was probably the neurotoxic agent anatoxin. All toxins in the different samples have earlier been shown to be produced by the cyanobacterial species used in the present study (for a review, see [17]). The mouse bioassay that preceded the genotoxicity testing clearly demonstrated that all four extracts were acutely toxic. The mice injected with sample 4 died within a few minutes and those injected with samples 1, 2, or 3 died within 1 h. Subsequent observations showed that samples 1, 2, and 3 caused a haemorrhagic shock, whereas sample 4 blocked nerve impulses. A possible disadvantage of using the in vivo micronucleus assay in animals and the in vitro micronucleus assay in human lymphocytes is that microcystins are known to accumulate in the liver. This occurs because there is an active uptake into parenchymal liver cells and the molecules do not readily cross all cell membranes [18–20]. In recent publications this accumulation has been shown to rely on certain organic anion-transporting polypeptides [21,22]. On the other hand, in studies showing accumulation of radiolabelled microcystins in the liver, other tissues were also shown to contain microcystins, e.g., lungs and gut, although to a much lower degree [23–26]. Furthermore, since uptake has been demonstrated at an early developmental stage in zebra-fish embryos [27] and even in aquatic plants [28,29], it indicates that also other uptake mechanisms are possible. In an in vitro study where the uptake and immunotoxic effects of microcystin-LR in lymphocytes were analysed, it was clear that also these cell types take up the toxin [30]. Microcystin-LR is soluble in water and thereby easily distributed in the blood system. Although the compound finally accumulates in the liver, it passes through different organs, e.g., the bone marrow. In the in vivo system the PCE (young erythrocytes) in peripheral blood are analysed about 40 h after the injection. This is the time interval from the last cell division of a certain cell population in the bone marrow – when the cells are exposed to the test compound – until the time when that same population reaches the peripheral blood system [10]. This would imply that in our study the exposed target cells were analysed at the appropriate time afterwards. The results from one study with extracts from cyanobacteria that contained high levels of microcystin-LR showed a weak but significant increase in the induction of micronuclei in mouse bonemarrow cells [31]. Based on these findings it was decided to use the sensitive flow cytometry-based micronucleus assay in vivo to further clarify these results. With this technique we have shown in earlier studies that it is possible to detect genotoxic effects at very low doses [12,32,33]. Even though several hundred thousands of PCE were analysed for each dose group, both after exposure to pure microcystin-LR and to the four samples of extracts, no genotoxic effect could be detected. There may be several explanations for the discrepancy in results found by us and by Ding et al. [31]. Different mouse strains as well as different extracts from different parts of the world, having different composition of toxin, may all be possible explanations. Furthermore, it is reasonable to believe that only one thousand PCE that was analysed from each mouse in the study of Ding et al. [31], is a number too small to give accurate results. In a study published by Gaudin et al. [34] it was demonstrated, using the comet assay, that after both intra peritoneal and oral administration of microcystin-LR in mice there are increased num-
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Table 1 The frequency of micro-nucleated polychromatic erythrocytes (fMNPCE) in male CBA mice exposed to different doses of microcystin-LR. The fMNPCE and PCE (%) were determined in peripheral blood 42 h after one intraperitoneal injection of microcystin-LR in different concentrations. Only the result for mice treated with colchicine (colch.) is significantly different from the control, p < 0.01 (Student’s t-test, two-tailed). Experiment 1, microcystin-LR (in vivo) Number of mice
Dose (g/kg bw)
PCE (%)
Number of PCE
Number of MNPCE
fMNPCE (‰)
3 2 2 2 2 2 2 3 2 2 2
0 10 20 30 34 38 42 46 50 55 colch.
2.6 2.6 2.7 2.7 2.5 2.5 2.4 2.1 2.1 3.4 0.6
734 856 440 402 431 072 423 660 504 704 472 834 485 248 601 472 477 733 477 801 78 948
975 624 579 534 616 577 615 831 569 615 463
1.33 1.44 1.34 1.26 1.22 1.22 1.27 1.38 1.19 1.29 5.8
Table 2 The frequency of micro-nucleated polychromatic erythrocytes (fMNPCE) in male CBA mice exposed to high doses of cyanobacterial extracts. The fMNPCE and PCE (%) were determined in peripheral blood 42 h after one intraperitoneal injection of samples of different cyanobacterial extracts. Only the result for mice treated with colchicine (colch.) is significantly different from the control, p < 0.01 (Student’s t-test, two-tailed). In the group of mice exposed to sample 1, three of the five mice died. Experiment 2, four different cyanobacteria extracts (in vivo) Number of mice
Sample number
Dose (mg/mouse)
PCE (%)
Number of PCE
Number of MNPCE
fMNPCE (‰) ± S.D.
5 2 5 5 5 3
Control 1 2 3 4 colch.
0 2 2 2 2
2.3 2.4 2.4 2.7 2.1 0.8
716 521 309 551 684 586 937 631 983 216 74 768
1189 380 1015 1891 1287 1187
1.59 ± 0.6 1.22 1.37 ± 0.4 2.00 ± 0.4 1.31 ± 0.2 3.8 ± 0.8
bers of DNA lesions in blood cells, e.g., strand breaks, labile sites and cross-links. The induction of DNA lesions was only observed at the highest dose, i.e., the effect was not dose-related. However, in that study the effect in the liver cells were more pronounced. In the present study we also exposed human lymphocytes to cyanobacterial extracts. Although 4000 bi-nucleated lymphocytes were analysed for each concentration (except one) no increased
micronucleus frequency was observed (Table 3). To our knowledge no other studies have reported micronucleus induction after in vitro exposure to cyanobacterial toxins or pure microcystin-LR when human lymphocytes (primary cell culture) were used. Using the comet assay on human lymphocytes, Mankiewicz et al. [35] found a correlation between the concentration of cyanobacterial toxins, containing different microcystins, and DNA lesions. The genotoxic-
Table 3 The frequency of micro-nucleated bi-nucleated lymphocytes (fBinMN) after in vitro exposure to different amounts of cyanobacterial extracts. The cell toxicity was calculated as the number of bi-nucleated lymphocytes among all lymphocytes (BinLymph). Peripheral blood was used from one donor, a healthy female, 55 years of age. Experiment 3, four different cyanobacteria extracts (in vitro) Sample number
Conc. (mg/ml media)
BinLymph (%)
BinLymph scored
BinMN scored
fBinMN (‰)
1 1 1 1 colch.
0 0.25 0.5 1 2 75 nM
46 47 40 33 35 3
4000 4000 4000 4000 4000 722
8 5 3 7 7 16
2 1.3 0.8 1.8 1.8 22
2 2 2 2 colch.
0 0.25 0.5 1 2 75 nM
34 19 23 19 17 3
8000 1500 4000 4000 4000 113
13 3 3 6 7 4
1.6 2 0.8 1.5 1.8 35
3 3 3 3 colch.
0 0.25 0.5 1 2 75 nM
21 25 20 43 16 1
4000 4000 4000 8000 4000 130
11 11 11 20 15 10
2.8 2.8 2.8 2.5 3.8 77
4 4 4 4 colch.
0 0.25 0.5 1 2 75 nM
31 17 7 22 21 1
4000 4000 4000 4000 4000 100
15 9 13 10 15 3
3.8 2.3 3.2 2.5 3.8 30
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ity of pure microcystin-LR in a human cell-line, lymphoblastoid TK6 cells, was reported by Zhan et al. [36]. They found an increased level of both micro-nucleated cells and TK mutation-induction at a high microcystin concentration. However, the increase of micronuclei was not dose-related, which may be due to the low number of cells scored: 1000 interphase cells for each treatment were analysed. In another cell-line, WIL2-NS lymphoblastoid cells, the authors showed by use of fluorescence in situ hybridisation that an increase of the micronucleus frequency was due to chromosome loss [37]. In this study the cells were exposed to the bacterial toxin cylindrospermopsin, produced by a number of cyanobacterium species, e.g., Cylindrospermopsis raciborskii. This toxin is a potent inhibitor of protein synthesis, but has a totally different chemical structure than microcystin. The impact of microcystin-LR on the mitotic spindle was demonstrated in Chinese hamster ovary (CHO-K1) cells. An increased concentration of the toxin caused defective chromosome separation resulting in polyploidy [38]. To clarify the possible mutagenic effect of cyanobacterial extracts and pure microcystin-LR, a handful of studies have been published using prokaryotic cells, i.e., the Salmonella mutation assay (Ames test). The results are conflicting, with genotoxic as well as non-genotoxic effects reported for cyanobacteria [31,39–41]. In these studies the presence of a metabolizing system did not influence the result. Other studies have demonstrated that the biotransformation of microcystin starts with the conjugation to gluthatione or to cysteine, which in turn decreases the binding capacity to, e.g., protein phosphatases and thus reduces the toxicity of microcystin. This has been shown in vivo and in vitro [42,43]. These findings were the reason why we did not include a metabolizing system in our in vitro experiments using human lymphocytes. To our knowledge no mutagenicity testing (gene mutation, chromosome mutation and aneuploidy) of cyanobacterial extract or pure microcystin-LR on liver cells has been reported. This may be due to the fact that there are few assays using these cell types with a sufficiently high sensitivity. On the other hand, increased levels of DNA strand breaks and oxidative damage-markers in liver cells after both in vitro and in vivo exposure to pure microcystin-LR or cyanobacterial extracts have been reported [31,34,44–48]. As mentioned earlier, it is sometimes stated that microcystins have a tumour-promoting effect. But if there is such an activity of microcystins, it is ascribed to their ability to bind to protein phosphatases, viz. PP1 and PP2A, thereby inhibiting their enzyme activity. Phosphorylation is one of the most important ways for an organism to change the structure and activity of a protein. Some specific defects in protein phosphorylation are known to cause human diseases, e.g., different forms of cancer, diabetes and certain inflammatory diseases [49]. Today there are several reports indicating that PP2A acts as a tumour-suppressor. Thus, inhibition of this enzyme may contribute to the development of cancer. Taking this information into account it seems likely that if microcystins are carcinogenic they act through a mechanism other than via genotoxicity. Our results support this conclusion. A linear relationship between dose and effect, which would be expected if a genotoxicity mechanism would be active, may thereby be excluded. Thus, the very low provisional guideline value of 1 g/l drinking-water as suggested by WHO may need to be re-evaluated. Conflict of interest statement None declared. Acknowledgements We would like to thank Madeleine Svensson for collecting information about the toxicity of cyanobacterial extracts, Ingalill
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Gadhasson for excellent technical assistance, Kristina Littmarck for skillfully analyzing the lymphocytes, AnnaMaria Thim and Siv Brostedt for the HPLC analyses. I thank Prof. emeritus Gösta Zetterberg and Maj Olausson for constructive criticism. References [1] WHO, Cyanobacterial toxins: microcystin-LR in drinking-water. Background document for preparation of WHO Guidelines for drinking-water quality, Geneva, World Health Organization (WHO/SDE/WSH/03.04/57), 2003. [2] I.R. Falconer, Tumor promotion and liver injury caused by oral consumption of cyanobacteria, Environ. Toxicol. Water Qual. 6 (1991) 177– 184. [3] I.R. Falconer, T.H. Buckley, Tumour promotion by Microcystis sp., a blue-green alga occurring in water supplies, Med. J. Aust. 150 (1989) 351. [4] R. Nishiwaki-Matsushima, T. Ohta, S. Nishiwaki, M. Suganuma, K. Kohyama, T. Ishikawa, W.W. Carmichael, H. Fujiki, Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR, J. Cancer Res. Clin. 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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Cyanobacterial extracts and microcystin-LR are inactive in the micronucleus assay in vivo and in vitro Lilianne Abramsson-Zetterberg a,∗ , Ulla Beckman Sundh a , Roland Mattsson b a b
The Swedish Food Administration, Box 622, 751 26 Uppsala, Sweden Department of Pathology and Wildlife, National Veterinary Institute, 751 89 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 2 November 2009 Received in revised form 22 January 2010 Accepted 29 January 2010 Available online 8 April 2010 Keywords: Microcystin-LR Cyanobacteria extract Micronuclei Flow cytometer
a b s t r a c t Cyanobacteria are sometimes widespread in lakes and can produce potent toxins, which can be dangerous for animals that drink the water, e.g. cattle and dogs. If the toxins are taken up by fish and other organisms in the food chain, or occur in drinking-water, they may pose a problem also for humans. Microcystin-LR, a hepatotoxic cyclic peptide, is one of the most frequently found cyanobacterial toxins. Data on the genotoxic potential of microcystin-LR and other cyanobacterial toxins are contradictory. Here we report results of the micronucleus assay carried out in vivo and in vitro with these toxins. To increase the sensitivity, we used the flow cytometry-based micronucleus assay in the mouse. In this study both pure microcystin-LR and cyanobacterial extracts originating from four different lakes in Sweden were analysed. Although doses up to near lethality were used and an average of 200,000 young erythrocytes, polychromatic erythrocytes, were analysed from each animal, no genotoxic effect was observed, nor could any effect be shown in the in vitro micronucleus study, using human lymphocytes. These results show that the low concentration of microcystins that now and then occur in drinkingwater does not increase the cancer risk through chromosome breaks or mal-distribution of chromosomes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cyanobacteria are sometimes widespread in lakes and can produce potent toxins. Several species of cyanobacteria produce different kinds of toxins. It is estimated that a large part of all water blooms are toxic. The production of cyanobacteria will increase in a warmer climate and when more nutrients leak into lakes from agricultural manuring. Animals, e.g., cattle and dogs that drink the water may be intoxicated. If the toxins are taken up by fish and other sea-food they may pose a problem also for humans. Contamination with cyanobacteria may also be a problem in drinking-water obtained from surface waters. Many bacterial toxins are eliminated in the refining process in the water plants. In spite of this, measurable amounts of microcystins may remain in the water. The exposure will be even higher for people who use this water for drinking without any filtering. The neurotoxic and/or hepatotoxic effects of the common toxins produced by some species of cyanobacteria are well known. The toxic effects are likely to have a threshold concentration below which no effect occurs. Thus the WHO suggests a tolerable daily intake (TDI) for microcystin-LR of 0.04 g/kg bw/day, a value
∗ Corresponding author. Tel.: +46 18 17 57 63; fax: +46 18 17 14 33. E-mail address: [email protected] (L. Abramsson-Zetterberg). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.04.001
assumed to safeguard against the neurotoxic and/or hepatotoxic effects [1]. However, if cyanobacterial toxins would be carcinogenic due to a genotoxic mechanism, even lower concentrations would be a potential problem. Convincing results about a carcinogenic effect of these substances have not been published so far. However, it is often stated in the literature that microcystins have tumour promoting and possibly also genotoxic or carcinogenic properties. In an early study, Falconer and co-worker [2,3] report indications of tumour-promoting activity when an extract of a naturally occurring toxic Microcystis bloom was given orally to mice initiated topically with dimethylbenzanthracene. There was an increase of the weight of skin tumours in these mice. The composition of the extract was, however, not defined and since microcystins are mainly taken up in the liver it would be surprising if these substances act as effective skin-tumour promoters. In a study by Nishiwaki-Matsushima et al. [4] the authors report tumourpromoting activity of microcystin-LR in livers of rats that had been initiated with diethylnitrosamine and partially hepatectomized. Increasing intraperitoneal doses of microcystin-LR corresponded with increasing numbers and areas of glutathione S-transferase (placental form)-positive foci in the livers, which was interpreted as an indication of tumour-promoting activity. Although this study is often cited as proof of the tumour-promoting activity of microcystins, it must be noted that glutathione S-transferase-positive foci are not an expression of tumour promotion per se. Other stud-
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ies have failed to show tumour-promoting or -initiating activity of microcystin-LR or of extracts of Microcystis, when applied orally [5–7]. However, when microcystin-LR was given via intraperitoneal injection during 28 weeks, the mice developed liver nodules [8]. Since there is uncertainty about the mechanism behind the interaction between microcystin-LR and the cells exposed, WHO has suggested an uncertainty factor of 1000 and a provisional guideline value of 1 g/l [1]. Published data about the genotoxic potential of microcystinLR and other cyanobacterial toxins are contradictory. When it can be clearly demonstrated that microcystin-LR is not mutagenic, the uncertainty factor may be lower. In order to contribute to the database concerning the suggested guideline concentration for microcystin-LR in drinking-water, we report results from some short-term tests using the micronucleus assay in vivo and in vitro. Both bacterial extracts and pure microcystin-LR were studied. To increase the sensitivity, we used the flow cytometry-based micronucleus assay in the mouse. 2. Material and methods This study comprises a total of three different experiments: two in vivo and one in vitro micronucleus study. In the first in vivo study (Experiment 1) the mice were exposed to different doses of microcystin-LR; in the second in vivo study (Experiment 2) the mice were exposed to extracts from four different samples of cyanobacteria (blue-green algae); in the third experiment (Experiment 3) the four cyanobacterial extracts were analysed in the in vitro micronucleus study with human lymphocytes. The extract samples originated from four lakes in Sweden, samples 1 and 3 from the southern and the northern part of the lake Mälaren (in the vicinity of Stockholm), sample 2 from the very southern part of Sweden, Skåne, and sample 4 from a lake in Småland (southern Sweden).
2.1. Collection, identification, and preparation of cyanobacterial samples The cyanobacteria were collected in the middle of the summer of 1985 (samples 1, 2, and 3) and 1987 (sample 4) from four different inland waters during the water-bloom period. Microscopic examination revealed that the dominating species in the respective samples were as follows: sample 1: 99% Planktothrix agardhii; sample 2: 80% Microcystis viridis, 20% Microcystis wesenbergi; sample 3: 99% Microcystis aeruginosa; sample 4: 85% Anabaena flos-aquae, 15% M. aeruginosa + Pl. agardhii + Gomphosphaeria sp. All samples were freeze-dried directly after examination and stored at −20 ◦ C. The toxins are stable and freeze-dried samples have been retested over time. For the toxicity test (bioassay) 250 mg of the freeze-dried sample were mixed in 5.0 ml NaCl (0.9%) for 15 min before being centrifuged at 10 min at 3200 × g. One milliliter of the supernatant was injected intraperitoneally in mice for the toxicity test. The mice were kept under observation and the symptoms, time of death, and the subsequent autopsy indicated the occurrence of toxins. Also a 10-fold lower dose, 0.1 ml of the toxin, resulted in an acute toxic effect. Prior to the genotoxicity tests, freeze-dried samples were extracted in the same way as for the tests of acute toxicity. The mice were given about half the lowest dose that resulted in death. The occurrence of different microcystins (Mic-LR, -YR, -RR) in cyanobacterial bloom samples was determined by high-performance liquid chromatography (HPLC) with diode-array detection. The analytical method used was based on a standard technique and an in-house method developed for freeze-dried algal samples [9]. HPLC analyses were performed on an Ace3 C18 reversed-phase 10-cm × 4.6-mm column with a Shimadzu LC10AD Vp chromatograph and Shimadzu diode-array detector SPD M10A Vp. Identification of the microcystin peaks was based on a comparison of retention times and absorbance spectra for samples and standard solutions of microcystins. Also a reference material of freeze-dried algae was analysed together with the samples. The concentrations of microcystins in the samples analysed were in the range 2–340 ng/mg.
2.2.2. Chemicals Microcystin-LR (CAS no. 101043-37-2), colchicine (CAS no. 64-86-8), and Hoechst 33342 (HO342) were purchased from Sigma–Aldrich, Sweden; Percoll was from Amersham Biosciences, Sweden; Fluothane from Astra, Sweden; and Thiazole orange (TO) from Molecular Probes, OR, USA. 2.2.3. Experimental design and sampling The genotoxic effects in vivo of microcystin-LR and of the four cyanobacterial extracts were determined using the flow cytometry-based micronucleus assay in mice. In both in vivo studies the animals were randomly divided in different groups. In all experiments the mice were injected intraperitoneally (i.p.) with a single dose of 10 l/g bw. Microcystin-LR and the cyanobacterial extracts were diluted in PBS just prior to injection. The positive control mice received injections of 1 mg/kg bw colchicine dissolved in PBS. In both in vivo studies (Experiments 1a and 1b) the mice were anaesthesized with Fluothane and blood samples were drawn from the orbital vein into heparinized tubes 42 h after injection. Directly after blood sampling the animals were killed by cervical dislocation. The sampling time was based on the known time interval between appearance of polychromatic erythrocytes (PCE) in the bone marrow and in peripheral blood [10]. In Experiment 1, which involved i.p. administration of microcystin-LR to male CBA mice, a total of 26 mice were used. They were given the following doses of microcystin-LR: 0, 10, 20, 30, 34, 38, 42, 46, 50, and 55 g/kg bw. All groups consisted of two mice except those given a dose of 0 and the highest doses, 46, 50, and 55 g/kg bw, and the positive control (colchicine), which all comprised three mice. Two out of the 26 mice died during the experiment. These mice had received the highest doses. Experiment 2, with i.p. administration of cyanobacterial extract to male CBA mice, involved 28 mice. For each of the four extracts, five mice were used. All five mice given the same extract received the same high dose. Results of the bioassay (see Section 2.1) where the acute toxicity of these four cyanobacterial extracts were tested, showed that a dose, two times used in this study, of 5 mg lyophilized algal cells/mouse was lethal. Taking these results into consideration we chose a dose of 2.0 mg of lyophilized algal cells/mouse. Three mice in the positive control group were given colchicine, and five mice constituted the negative control group and received PBS. 2.2.4. Purification, fixation and staining of erythrocytes The methods for purification, fixation and staining of erythrocytes have been published previously [11,12]. Briefly, 5 l of whole blood was layered on a 65% Percoll gradient and centrifuged. The supernatant was carefully removed and the pellet was re-suspended in PBS and fixed in glutaraldehyde according to a method described by Hayashi et al. [13]. The samples were coded and stored at 4 ◦ C for a few days. A staining solution, containing the fluorescent dyes HO342 (DNA-dye) and TO (RNA-dye) in PBS, was added to the fixed cells. The staining was performed 1 day prior to analysis. 2.2.5. Flow-cytometric analysis and the determination of micronucleus frequency For the analysis a FACSVantage SE flow cytometer (Becton-Dickinson Immunocytometry Systems, Sunnyvale, CA) was used according to a method described elsewhere [11,12]. The cells were automatically analysed when they, one by one, passed through two laser beams (350 and 488 nm). Information about size and structure was collected and used to exclude remaining nucleated cells as well as debris in the sample from further analysis. DNA- and RNA-content was detected as fluorescence from the two dyes HO342 and TO, respectively. RNA content, measured as the signal from the TO dye, was used to distinguish between young and old erythrocytes, PCE and NCE (poly- and normochromatic erythrocytes), respectively. The ratio of young to old cells gives an indication of cell proliferation (%PCE). This analysis was based on the information from about 20,000 cells per sample. Furthermore, since erythrocytes normally do not contain any DNA, a signal from the HO342 dye implies that it contains a micronucleus. Limiting the analysis to only PCE enabled us to calculate the frequency of micro-nucleated PCE. About 200,000 PCE were scored per animal. All analyses were performed using CellQuest software (Becton-Dickinson). Scatter plots of the information about DNA content vs RNA content were displayed for each analysed sample. Regions were defined for NCE, PCE, and MNPCE (micro-nucleated PCE), respectively. On the basis of this, the number of events in each region was determined. Frequencies of PCE and MNPCE were calculated. 2.3. Micronucleus assay in vitro in human lymphocytes (Experiment 3)
2.2. Micronucleus assay in vivo in mice (Experiments 1 and 2) 2.2.1. Animals Male CBA mice, 6–7 weeks old, weighing approximately 25 g, were obtained from Scanbur AB, Sollentuna, Sweden. The animals were housed at the Swedish National Food Administration in a 12 h light/12 h dark cycle with free access to solid food and tap water. All mice were acclimatised 1 week before treatment. The experiment was reviewed and approved by Uppsala Ethical Committee on Animal Experiments, application C295/6.
2.3.1. Chemicals F-10 HAM’s medium and fetal calf serum were from HyClone USA; penicillin/streptomycin (PEST) and l-glutamine from Bio Whittaker Europe; phytohaemagglutinin (PHA), cytochalasin B, and colchicine (CAS no. 64-86-8) from Sigma–Aldrich, Sweden. 2.3.2. Experimental design and treatment One experiment was performed with blood from a healthy non-smoking female, 55 years old. Whole blood cultures were treated with the four cyanobacterial
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Fig. 1. Results of the analysis by flow cytometry of polychromatic erythrocytes (PCE) from peripheral blood of male CBA mice given single injections (i.p.) with different amounts of microcystin-LR. Blood samples were taken 42 h after injection. Each data point is the mean value of fMNPCE for two mice, except for the mice given a dose of 0 and 46 g/kg bw, where each of the data points represents three mice. Two mice in each of the highest dose groups died during the experiment. There is no significant dose-response trend (see Table 1). The fMNPCE (frequency of micro-nucleated polychromatic erythrocytes in peripheral blood) for the mice given colchicine (1 mg/kg bw) was significantly different from the control level (p < 0.01).
extracts dissolved in PBS. Concentrations of 0, 0.25, 0.5, 1.0, and 2.0 mg extract of freeze-dried cyanobacteria per ml cell culture were used for each of the four extracts, respectively. Colchicine, final concentration 75 nM, was used as a positive control. The experiments were carried out according to the method described by Fenech and Morley [14] and refined by Migliore et al. [15]. Briefly, whole blood was cultured in F-10 HAM’s medium supplemented with 12% fetal calf serum, 0.5% l-glutamine, 2% PEST, and 1.2% PHA for 72 h. The cultures were kept in a humidified environment with 5% CO2 at 37 ◦ C. After 24 h of incubation, the different agents were added according to the experimental setup. After 44 h cytochalasin B was added to the cultures to a final concentration of 6 g/ml. The incubation continued until 72 h after initiation. The cultures were then centrifuged and the supernatant was removed. Erythrocytes were lysed by addition of KCl (0.075 M). After 3 min, prefixative (methanol/acetic acid 3:1, v/v) was added. The cells were rinsed and fixed in methanol and methanol/acetic acid (5:1, v/v) and spread on clean, cold, wetted glass slides. After drying, the cells were stained with 3% Giemsa dye. 2.3.3. Scoring All slides were randomised and coded prior to analysis. The scoring was performed in accordance with Fenech [16] using a Leica Ortholux II microscope. About 4000 bi-nucleated lymphocytes (cells that have undergone one mitotic division during the exposure) from each culture were scored for the presence of micronuclei. As a measure of cell division the proportion of bi-nucleated cells (% BinLymph) among the total of viable cells was calculated. About 600 cells per culture were scored for this purpose.
3. Results and discussion In this study the frequency of micro-nucleated cells was analysed in vivo and in vitro after exposure to both pure microcystin-LR and cyanobacterial extracts from four different lakes in Sweden. All four extracts showed acute toxicity in the mouse bioassay (see Section 2.1). None of the animals given different doses of microcystin-LR or the cyanobacterial extracts, showed an increased frequency of micro-nucleated polychromatic erythrocytes in the peripheral blood, fMNPCE (see Tables 1 and 2 and Fig. 1). Neither did the percentage of polychromatic erythrocytes, %PCE, reveal any changes in the cell proliferation (Tables 1 and 2). In the in vitro assay, an increased concentration of the extracts did not result in a concomitantly higher frequency of micro-nucleated bi-nucleated human lymphocytes, fBinMN (Table 3). In both the in vivo and in vitro studies sufficiently high doses and concentrations were analysed. At the highest doses of microcystin-LR or of the extract, an adverse effect was noticed, i.e., some mice died and some showed signs of distress (fur condition, wakefulness, etc.). In addition, the concentrations used in the in vitro study were limited because it was not possible to dissolve higher amounts of the extracts. Only the
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mice and cells exposed to the positive control, colchicine, showed a significant increase in the mean fMNPCE or fBinMN. The HPLC analysis showed that three of the four extracts contained different kinds of microcystin. Three microcystins (Mic) could be quantified, i.e., Mic-RR, -LR, and -YR. The analysis showed that sample 1 contained about 70% Mic-LR, 30% Mic-YR, <1% MicRR; sample 2: 54% Mic-RR, 40% Mic-LR, 6% Mic-YR; sample 3: 40% Mic-RR, 60% Mic-LR, <1% Mic-YR. Sample 4 mainly contained toxins other than microcystins. According to the algal analysis and from observation of the mice, the major toxin was probably the neurotoxic agent anatoxin. All toxins in the different samples have earlier been shown to be produced by the cyanobacterial species used in the present study (for a review, see [17]). The mouse bioassay that preceded the genotoxicity testing clearly demonstrated that all four extracts were acutely toxic. The mice injected with sample 4 died within a few minutes and those injected with samples 1, 2, or 3 died within 1 h. Subsequent observations showed that samples 1, 2, and 3 caused a haemorrhagic shock, whereas sample 4 blocked nerve impulses. A possible disadvantage of using the in vivo micronucleus assay in animals and the in vitro micronucleus assay in human lymphocytes is that microcystins are known to accumulate in the liver. This occurs because there is an active uptake into parenchymal liver cells and the molecules do not readily cross all cell membranes [18–20]. In recent publications this accumulation has been shown to rely on certain organic anion-transporting polypeptides [21,22]. On the other hand, in studies showing accumulation of radiolabelled microcystins in the liver, other tissues were also shown to contain microcystins, e.g., lungs and gut, although to a much lower degree [23–26]. Furthermore, since uptake has been demonstrated at an early developmental stage in zebra-fish embryos [27] and even in aquatic plants [28,29], it indicates that also other uptake mechanisms are possible. In an in vitro study where the uptake and immunotoxic effects of microcystin-LR in lymphocytes were analysed, it was clear that also these cell types take up the toxin [30]. Microcystin-LR is soluble in water and thereby easily distributed in the blood system. Although the compound finally accumulates in the liver, it passes through different organs, e.g., the bone marrow. In the in vivo system the PCE (young erythrocytes) in peripheral blood are analysed about 40 h after the injection. This is the time interval from the last cell division of a certain cell population in the bone marrow – when the cells are exposed to the test compound – until the time when that same population reaches the peripheral blood system [10]. This would imply that in our study the exposed target cells were analysed at the appropriate time afterwards. The results from one study with extracts from cyanobacteria that contained high levels of microcystin-LR showed a weak but significant increase in the induction of micronuclei in mouse bonemarrow cells [31]. Based on these findings it was decided to use the sensitive flow cytometry-based micronucleus assay in vivo to further clarify these results. With this technique we have shown in earlier studies that it is possible to detect genotoxic effects at very low doses [12,32,33]. Even though several hundred thousands of PCE were analysed for each dose group, both after exposure to pure microcystin-LR and to the four samples of extracts, no genotoxic effect could be detected. There may be several explanations for the discrepancy in results found by us and by Ding et al. [31]. Different mouse strains as well as different extracts from different parts of the world, having different composition of toxin, may all be possible explanations. Furthermore, it is reasonable to believe that only one thousand PCE that was analysed from each mouse in the study of Ding et al. [31], is a number too small to give accurate results. In a study published by Gaudin et al. [34] it was demonstrated, using the comet assay, that after both intra peritoneal and oral administration of microcystin-LR in mice there are increased num-
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Table 1 The frequency of micro-nucleated polychromatic erythrocytes (fMNPCE) in male CBA mice exposed to different doses of microcystin-LR. The fMNPCE and PCE (%) were determined in peripheral blood 42 h after one intraperitoneal injection of microcystin-LR in different concentrations. Only the result for mice treated with colchicine (colch.) is significantly different from the control, p < 0.01 (Student’s t-test, two-tailed). Experiment 1, microcystin-LR (in vivo) Number of mice
Dose (g/kg bw)
PCE (%)
Number of PCE
Number of MNPCE
fMNPCE (‰)
3 2 2 2 2 2 2 3 2 2 2
0 10 20 30 34 38 42 46 50 55 colch.
2.6 2.6 2.7 2.7 2.5 2.5 2.4 2.1 2.1 3.4 0.6
734 856 440 402 431 072 423 660 504 704 472 834 485 248 601 472 477 733 477 801 78 948
975 624 579 534 616 577 615 831 569 615 463
1.33 1.44 1.34 1.26 1.22 1.22 1.27 1.38 1.19 1.29 5.8
Table 2 The frequency of micro-nucleated polychromatic erythrocytes (fMNPCE) in male CBA mice exposed to high doses of cyanobacterial extracts. The fMNPCE and PCE (%) were determined in peripheral blood 42 h after one intraperitoneal injection of samples of different cyanobacterial extracts. Only the result for mice treated with colchicine (colch.) is significantly different from the control, p < 0.01 (Student’s t-test, two-tailed). In the group of mice exposed to sample 1, three of the five mice died. Experiment 2, four different cyanobacteria extracts (in vivo) Number of mice
Sample number
Dose (mg/mouse)
PCE (%)
Number of PCE
Number of MNPCE
fMNPCE (‰) ± S.D.
5 2 5 5 5 3
Control 1 2 3 4 colch.
0 2 2 2 2
2.3 2.4 2.4 2.7 2.1 0.8
716 521 309 551 684 586 937 631 983 216 74 768
1189 380 1015 1891 1287 1187
1.59 ± 0.6 1.22 1.37 ± 0.4 2.00 ± 0.4 1.31 ± 0.2 3.8 ± 0.8
bers of DNA lesions in blood cells, e.g., strand breaks, labile sites and cross-links. The induction of DNA lesions was only observed at the highest dose, i.e., the effect was not dose-related. However, in that study the effect in the liver cells were more pronounced. In the present study we also exposed human lymphocytes to cyanobacterial extracts. Although 4000 bi-nucleated lymphocytes were analysed for each concentration (except one) no increased
micronucleus frequency was observed (Table 3). To our knowledge no other studies have reported micronucleus induction after in vitro exposure to cyanobacterial toxins or pure microcystin-LR when human lymphocytes (primary cell culture) were used. Using the comet assay on human lymphocytes, Mankiewicz et al. [35] found a correlation between the concentration of cyanobacterial toxins, containing different microcystins, and DNA lesions. The genotoxic-
Table 3 The frequency of micro-nucleated bi-nucleated lymphocytes (fBinMN) after in vitro exposure to different amounts of cyanobacterial extracts. The cell toxicity was calculated as the number of bi-nucleated lymphocytes among all lymphocytes (BinLymph). Peripheral blood was used from one donor, a healthy female, 55 years of age. Experiment 3, four different cyanobacteria extracts (in vitro) Sample number
Conc. (mg/ml media)
BinLymph (%)
BinLymph scored
BinMN scored
fBinMN (‰)
1 1 1 1 colch.
0 0.25 0.5 1 2 75 nM
46 47 40 33 35 3
4000 4000 4000 4000 4000 722
8 5 3 7 7 16
2 1.3 0.8 1.8 1.8 22
2 2 2 2 colch.
0 0.25 0.5 1 2 75 nM
34 19 23 19 17 3
8000 1500 4000 4000 4000 113
13 3 3 6 7 4
1.6 2 0.8 1.5 1.8 35
3 3 3 3 colch.
0 0.25 0.5 1 2 75 nM
21 25 20 43 16 1
4000 4000 4000 8000 4000 130
11 11 11 20 15 10
2.8 2.8 2.8 2.5 3.8 77
4 4 4 4 colch.
0 0.25 0.5 1 2 75 nM
31 17 7 22 21 1
4000 4000 4000 4000 4000 100
15 9 13 10 15 3
3.8 2.3 3.2 2.5 3.8 30
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ity of pure microcystin-LR in a human cell-line, lymphoblastoid TK6 cells, was reported by Zhan et al. [36]. They found an increased level of both micro-nucleated cells and TK mutation-induction at a high microcystin concentration. However, the increase of micronuclei was not dose-related, which may be due to the low number of cells scored: 1000 interphase cells for each treatment were analysed. In another cell-line, WIL2-NS lymphoblastoid cells, the authors showed by use of fluorescence in situ hybridisation that an increase of the micronucleus frequency was due to chromosome loss [37]. In this study the cells were exposed to the bacterial toxin cylindrospermopsin, produced by a number of cyanobacterium species, e.g., Cylindrospermopsis raciborskii. This toxin is a potent inhibitor of protein synthesis, but has a totally different chemical structure than microcystin. The impact of microcystin-LR on the mitotic spindle was demonstrated in Chinese hamster ovary (CHO-K1) cells. An increased concentration of the toxin caused defective chromosome separation resulting in polyploidy [38]. To clarify the possible mutagenic effect of cyanobacterial extracts and pure microcystin-LR, a handful of studies have been published using prokaryotic cells, i.e., the Salmonella mutation assay (Ames test). The results are conflicting, with genotoxic as well as non-genotoxic effects reported for cyanobacteria [31,39–41]. In these studies the presence of a metabolizing system did not influence the result. Other studies have demonstrated that the biotransformation of microcystin starts with the conjugation to gluthatione or to cysteine, which in turn decreases the binding capacity to, e.g., protein phosphatases and thus reduces the toxicity of microcystin. This has been shown in vivo and in vitro [42,43]. These findings were the reason why we did not include a metabolizing system in our in vitro experiments using human lymphocytes. To our knowledge no mutagenicity testing (gene mutation, chromosome mutation and aneuploidy) of cyanobacterial extract or pure microcystin-LR on liver cells has been reported. This may be due to the fact that there are few assays using these cell types with a sufficiently high sensitivity. On the other hand, increased levels of DNA strand breaks and oxidative damage-markers in liver cells after both in vitro and in vivo exposure to pure microcystin-LR or cyanobacterial extracts have been reported [31,34,44–48]. As mentioned earlier, it is sometimes stated that microcystins have a tumour-promoting effect. But if there is such an activity of microcystins, it is ascribed to their ability to bind to protein phosphatases, viz. PP1 and PP2A, thereby inhibiting their enzyme activity. Phosphorylation is one of the most important ways for an organism to change the structure and activity of a protein. Some specific defects in protein phosphorylation are known to cause human diseases, e.g., different forms of cancer, diabetes and certain inflammatory diseases [49]. Today there are several reports indicating that PP2A acts as a tumour-suppressor. Thus, inhibition of this enzyme may contribute to the development of cancer. Taking this information into account it seems likely that if microcystins are carcinogenic they act through a mechanism other than via genotoxicity. Our results support this conclusion. A linear relationship between dose and effect, which would be expected if a genotoxicity mechanism would be active, may thereby be excluded. Thus, the very low provisional guideline value of 1 g/l drinking-water as suggested by WHO may need to be re-evaluated. Conflict of interest statement None declared. Acknowledgements We would like to thank Madeleine Svensson for collecting information about the toxicity of cyanobacterial extracts, Ingalill
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