Effects induced by pure MCLR and toxic
Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with contaminated water . This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant . MCs are mainly known for their hepatotoxicity due to their inhibitory activity of serine/threonine phosphatases PP1 and PP2A . This inhibition interferes with hepatocyte homeostasis and structure, leading to the collapse of liver tissue organization, liver necrosis and hemorrhage (Figure 1), which can culminate, in severe cases, in the death of the intoxicated individuals [3, 4].
It has been reported that microcystins cross cell membranes through the transmembrane solute carriers transport family OAPT (Organic Anion Polypeptide Transporters), in particular the OATP1B1, OATP1B2, OATP1B3 and OATP1A2 [7, 8]. They are responsible for the sodium-independent uptake of large amphipathic endogenous and exogenous organic anions into cells and across the blood-brain barrier [9, 10]. The knowledge on the mechanism(s) of OATP-mediated transport is still scarce  however, the available information suggests that, in a general way, OATPs act as organic anion exchangers , functioning in a rocker-switch type of mechanism and translocating the substrate through a central positively charged pore .
Some OATPs are expressed ubiquitously, whereas others are expressed in a tissue-specific way . In fact, the organotropism of MC is due to the selective uptake of microcystins by the OATPs that are primarily expressed in liver , such as those mentioned above. For this reason, the study of the toxicological properties of microcystins has been conducted mainly in liver cells
Established cell lines have been considered, for a long time, as unsuitable to study the toxic effects of MCs. This was due to the observation that, comparing with primary cell lines, high amounts of MCs were required to elicit toxicity in permanent cell lines [17, 18, 19]. A proposed explanation was the fact that established cell lines lose their OATPs, which render them unable to uptake the toxin [19, 20]. Despite this fact, an increasing number of studies have demonstrated that MCs clearly induce toxic effects on several mammalian cell lines, in particular in the human hepatoma HepG2 cell line [21-24]. Indeed, it was already demonstrated that OATP transport system is preserved in the HepG2 cells .
When we started our work on MCLR toxicity in mammalian cell lines we evaluated the effects of
To our knowledge no previous study reported the expression of OATPs in Vero cell line, neither if an alternative transport system is involved in the microcystin uptake by kidney cells. The elucidation of this issue would be obviously an important contribution to the knowledge of microcystins toxicokinetics in the kidney and, consequently, an important stimulus to the investigation of microcystins toxicity in the kidneys.
The studies of MCs effects on cell lines often lead to contradictory results, given the fact that distinct MC toxicity endpoints (mainly cytotoxicity and genotoxicity) have been studied in diverse cell lines (and cell clones) under distinct exposure conditions (different doses-ranges, time of exposure, MCs variants, etc). In our work with Vero-E6 cells we tested MCLR (both pure toxin and from cyanobacterial extracts of
2. Effects of MCLR in Vero-E6 cell line
2.1. Cell viability
In our studies with Vero-E6 cell line we have consistently observed that MCLR induces a concentration-dependent decrease of cell viability [26, 27, 29, 30]. This was achieved using distinct cell viability assays (MTT, Neutral Red and LDH release) and distinct toxin sources (commercially available MCLR and
2.2. Cellular organelles
In Vero-E6 cell line, exposure to MCLR (within the micromolar concentrations range) affected several cellular organelles in a concentration-dependent form (Table 1, Figure 3). In our articles, we proposed that at lower MCLR concentrations, autophagy is triggered as a survival mechanism of Vero cells, in an attempt to eliminate the toxin or/and the MCLR-induced cellular damages [29, 30]. Also, we reported the vacuolization of the Golgi apparatus and cytoplasm, effects also previously described in MCLR-exposed hepatocytes [17, 18]. The disorganization of the microfilaments and microtubules, which is one of the most commonly described cytotoxic effects of MCLR in hepatic cells [34-36], was also triggered by MCLR in Vero cells [29, 30]. The observation that MCLR induced the destructuring of the endoplasmic reticulum (ER) suggested that this organelle is involved in MCLR toxicity in Vero cell line [29, 30]. The involvement of the ER in MCLR-mediated toxicity was also previously reported in mouse kidney and liver . At higher concentrations, MCLR induced the disruption of the plasma membrane, lysosomes and mitochondria of Vero cells [29, 30]. So far, studies that have used other cell types proposed mitochondria as a major target of MCLR toxicity [38-42]. However, studies with Vero cells show that mitochondria, although involved in MCLR-induced toxicity, may not be the pivotal intracellular target of the toxin [29, 30].
Based on its tumour promoter activity MCLR is classified by the International Agency for Research on Cancer as a potential human carcinogen (class 2B) . In addition, some epidemiologic studies have associated the increase of human hepatocarcinoma [44, 45] and colorectal  cancers with the ingestion of water frequently contaminated with microcystins. However it is still unclear if, besides a tumor promoter, MCLR can also act as a tumor initiator. In fact, the potential genotoxicity of MCLR is still a matter of some controversy within the scientific community, with several authors reporting apparently contradictory results.
The hypothesis of MCLR being a genotoxic compound was mainly supported by the evidence that this toxin induces DNA damage in liver cells
Chromosome damage (aneugenesis/clastogenesis) has been suggested for microcystins by an increase of the micronucleus (MN) frequency in mouse erythrocytes  and in the human TK6 cell line . However, no effect on the micronucleus frequency has been reported for other cell types [50, 54-56] neither chromosome aberrations have been described so far [54, 57].
Some authors have also proposed that MCLR might interfere with DNA repair processes, namely the repair of gamma radiation and ultra-violet-induced DNA damage [52, 54, 57], thus increasing the genotoxic potency of those agents and contributing to their carcinogenesis.
|25 – 200 µM (52-82%)||22-175 µM (55-58%)||MTT assay||
|_||30 µM - 150 µM (35-67%)||MTT assay||
|_||30 µM - 150 µM (26-79%)||Neutral Red assay||
|50 -100 µM (48-66%)||50 -100 µM (80-82%)||Neutral Red assay||
|200 µM (55%)||88-175 µM (38-29%)||LDH release||
||20-50 µM (13-27%)|
||12-100 µM (10-25%)|
||Within micromolar range||20 and 40 µM (3.4 and 4.1 fold)||Micronucleus assay||
||5-5000 nM (2.8–4 fold)||5-5000 nM (1.7–3.7 fold)||Western Blot||
||1– 10 nM (1.5-2.1 fold)||-||BrdU incorporation assay|
In our early studies with the Vero-E6 cell line we demonstrated that cyanobacterial extracts from a MCLR-producer cyanobacterial strain increased the frequency of micronuclei at non-cytotoxic concentrations . Afterwards, we confirmed this observation using pure MCLR [58, submitted for publication]. In this study, we found that MCLR induces the micronucleation of Vero-E6 and human hepatoma HepG2 cell lines. In order to disclose the mechanism underlying micronuclei formation we used the centromere labelling Fluorescent in situ Hybridization technique in HepG2 exposed cells to MCLR (since there are no commercial centomere probes for monkey cells) and we observed that MCLR induced both centromere-positive and centromere-negative micronuclei . Our data, together with those from other works, suggests that MCLR genotoxicity occurs indirectly by both clastogenic and aneugenic mechanisms: the first, possibly through oxidative stress; the second, perhaps through damages on the mitotic spindle, induced by the inhibition of PP1/PP2A. Since clastogenesis and aneugenesis have been associated with human cancer development  it can be hypothesized that both underlie the carcinogenic activity of MCLR. Moreover, the confirmation of genotoxic activity of MCLR
In figure 4 we summarize the proposed effects/mechanisms of MCLR genotoxicity, based on the previous reports from other authors and our own contribution.
2.4. Cell proliferation
It is generally assumed that MCLR is a potent tumor promoter. This assumption is based on rodent carcinogenicity studies which revealed that MCLR is able to induce cellular transformation of rat liver  and mouse skin  of animals previously exposed to a genotoxic agent. However, the mechanisms underlying MCLR-induced tumor promotion are still unknown. It has been suggested that this activity is mediated by the inhibition of serine/threonine phosphatase PP1 and PP2A, given their role on the regulation of cellular division and proliferation, namely through the activation of Mitogen-Activated Protein Kinases (MAPK) . MAPKs are involved in signaling pathways that regulate many cellular processes through phosphorylation cascades, in particular the Ras-Raf-MEK1/2-ERK1/2 cascade, with a key role in cellular proliferation and being regulated by several types of phosphatases including the serine/threonine phosphatases PP1 and PP2A . The Ras-Raf-MEK1/2-ERK1/2 cascade is activated by growth factors and mitogenic agents (Figure 5). They bind to tyrosine kinase membrane receptors (RTK), which activate the membrane G-protein GTPase, the recruitment and activation of Raf protein and the subsequent phosphorylation cascade of ERK1/2 pathway . The activated (phosphorylated) forms of ERK1/2 are translocated to the cell nucleus, inducing the activation of transcription factors such as c-Fos and c-Jun thus triggering cell proliferation . The fact that Ras-Raf-MEK-ERK cascade is regulated by several types of phosphatases including the protein serine/threonine phosphatases PP2A [64, 66] supports the hypothesis that, by inhibiting PP2A, MCLR deregulates the ERK1/2 pathway and promotes cell proliferation (Figure 5).
Few studies support this hypothesis: (1) Li et al  reported the activation of proto-oncogenes c-jun, c-fos and c-myc by a cyanobacterial extract containing microcystins in rat liver, kidney and testis; (2) Zhu et al  demonstrated that MCLR induces the transformation of immortalized colorectal crypt cells through the constitutive activation of AKT and MAPK (p38 and JNK) cascades. Our team has evaluated the effect of MCLR in Vero-E6 cell line proliferation through the BrdU incorporation assay that evaluates the G1/S transition in cell cycle . We showed that MCLR (1 to 10 nM) induces a significant increase in Vero cells proliferation with a maximum of 2.2 fold increase at 5 nM . We further analyzed the expression of MAPK (ERK1/2, JNK and p38) by Western-blot and concluded that MCLR stimulates Vero cells proliferation by the activation of the ERK1/2 signaling pathway . These results emphasize the importance to confirm the impact of MCLR on tumor promotion
3. Comparison of MCLR-induced toxicity in kidney cell lines
The effects of microcystins in kidney cell lines, namely in Vero cells, have been barely evaluated. Thompson et al  reported that cyanobacteria extracts containing up to 10 µM of MCLR did not interfere with the morphology and LDH release of Vero cells. Chong et al.  also did not found changes in Vero cells viability (evaluated by the MTT assay) after exposure to concentrations up to 37.5 µM of pure MCLR during 24–96 h. Grabow et al.  described cytopathogenic effects (rounding and disintegration of cells) of Vero cells induced by
Additionally, in a previous study we also observed that MCLR induce cytotoxic effects in the Madin-Darbin canine kidney cell line (MDCK – ATCC CCL-34) . However, the sensitivity of this cell line was lower than that of Vero cells. In fact, while significant reduction of Vero cells viability occurs above 25 µM of MCLR (Table 1), only 30% decrease in MDCK cell viability was observed after exposure to 100 µM of MCLR, using the Neutral Red Assay .
Effects of MCLR on cell morphology and ultrastructure were previously evaluated in the rat renal epithelial cell line NRK-52E (ATCC-CRL 1571) in studies developed in 1990’s decade. Wickstrom et al.  found that MCLR affects the cytoskeleton components namely the microtubules, intermediate filaments and microfilaments in a similar way to that observed in hepatocytes. However, the renal cell line required a 100-fold higher concentration (more than 100 µM) and prolonged time of exposure comparing to primary hepatocytes . Reports from Khan et al. [17, 74] also demonstrated the collapse and condensation of cystoskeleton elements induced by MCLR (133 µM) on NRK-52E cell line.
The differences reported in the above mentioned distinct studies might be explained by differences in experimental design, such as the use of toxins from different sources (pure or crude extracts), applied in different dosages and tested by different endpoints of toxicity. Besides, the use of different clones of Vero cells (often not mentioned in the papers) may also justify the distinct sensitivities observed among several authors. Further studies would also be required to conclude if the effects of MCLR on kidney cells could be species-dependent.
Overall, the cytotoxic, morphological and ultrastructural effects of MCLR on Vero-E6 cell line reported by us are quite similar to those reported for other cell lines. However, we observed these effects at lower concentrations, which might suggest an eventual higher sensitivity of Vero-E6 cell line comparing to other kidney cell lines.
4. Is Vero-E6 cell line a suitable model to study toxicological properties of microcystins?
The assessment of kidney injury/dysfunction
Vero-E6 monkey kidney cell line has been widely used on toxicology, virology and pharmacology research, as well as, on the production of vaccines and diagnostic reagents . In particular, these cells have been used as model for assays to evaluate the toxicity of compounds of different nature, either chemical or microbial toxins. The chemical substances tested include carbamazepine ; triclosan ; lead nitrate ; pentachlorophenol and rotenone , where the Vero cell line revealed to be one of the most sensitive model used in these studies. These cells have also been validated as a cellular model for other microbial toxins such as diphtheria toxin, a polypeptide with 535 a.a.  and Shiga-like toxins, a protein of enterohemorrhagic
In our studies on the effects of MCLR on Vero-E6 cell line we did not evaluate any specific nephrotoxicity marker. Instead, we evaluated the basal toxicity of MCLR, that is, the effects that might be common to all cell types .
Using diverse methodologies including standard methods to evaluate cytotoxicity (Neutral Red, MTT and Lactate Dehydrogenase release) and genotoxicity (Micronucleus and Comet assay) we observed that MCLR induces a multiplicity of effects on Vero-E6 cells at distinct levels: cellular morphology/ultrastructure, cell viability/death, MAPK expression and genotoxicity (as referred in section 2). The type and extension of these effects were highly dependent of toxin concentration and, generally, a dose-response relation could be established: for a dose range of 1-10 nM, MCLR stimulates cell proliferation through the activation of the mitogen activated protein kinase ERK1/2 signalling pathway; however, within the μM range, MCLR triggers a variety of effects in almost all cell compartments, from genotoxicity (induction of micronuclei) and autophagy to apoptotic and necrotic cell death. Therefore, MCLR induces a dual effect on the Vero-E6 cell line: at low doses it stimulates the cell growth but at high doses it induces a decrease in viability and cell death (Figure 6).
This duality of low-dose growth stimulation
Another aspect we would like to underline is that we can attribute with high certainty the observed responses of Vero-E6 cell line to MCLR exposure. The results obtained with toxic cyanobacterial extracts are often questioned due to the uncertainty of cyanobacteria extracts composition and the putative interference between cyanotoxins and other cyanobacterial bioactive compounds . In our studies, we tested two sources of MCLR: cyanobacterial extracts from MCLR-producers (strains of
Usually it is assumed that high amounts of MCLR are required to elicit any effect on cell lines comparing to primary cells . This is true, but it also depends on the effects that are being evaluated. In fact, cytotoxic MCLR concentrations for Vero cells were found within the range of micromolar (µg/mL). However, the lowest concentration that elicited an effect (cell proliferation) on Vero-E6 cell line was 1 nM (µg/L) . Many of the effects induced by MCLR in Vero-E6 cell line were quite similar to those induced in the human hepatoma cell line (HepG2) at similar dose-ranges. This applies to our data on MCLR genotoxicity on distinct cell models  as well as on reported data from other authors [22, 47]. In fact, HepG2 cell line has been considered a suitable model to evaluate the effects of MCLR on liver-derived cells, which similarly might be applicable to Vero-E6 cell line regarding kidney-derived cells.
Considering all these aspects, we propose that Vero-E6 cell line is an appropriate
5. Concluding remarks
Despite the gaps in knowledge regarding the toxicological properties of microcystins, an increasing number of recent publications emphasize the need to carefully evaluate their effects on other organs besides the liver, particularly their carcinogenicity. The adverse effects of MCLR in distinct organs is an important issue for risk assessment, because the guideline value for MC in drinking water (1 nM) is still a provisional value, based on limited toxicological data .
The exposure to low doses of MCLR corresponds to the most realistic kidney intoxication scenario, considering that it is not the main target organ of this toxin. However, the role of kidneys in toxin elimination might lead to the exposure of kidney cells to a low internal dose that can be biologically effective in the induction of nephrotoxic effects. Besides, the chronic adverse effects of microcystins on kidneys, specially the potential tumorigenic/carcinogenic effects, may assume a particular importance in populations exposed to water persistently contaminated with toxic cyanobacteria.
Although further studies will be required to recognize the epithelial monkey kidney-derived Vero-E6 cell line as a nephrotoxicity model, we propose that Vero-E6 cell line constitutes a valuable in vitro model to evaluate the basal toxicity of MCLR and to study the mechanisms of MCLR toxicity.
In figure 7 we summarize the effects induced by MCLR on Vero-E6 cell line.
Funari E, Testai E. Human health risk assessment related to cyanotoxins exposure. Critical Rev Toxicol 2008;38:97-125.
Yoshizawa I, Matsushima R, Watanabe MF, Harada H, Ichihara A, Carmichael WW, Fujiki H. Inhibition of protein phosphatases by microcystis and nodularin associated with hepatotoxicity. J Cancer Res Clin Oncol. 1990;116:609-614.
Falconer IR, Yeung DS. Cytoskeletal changes in hepatocytes induced by microcystis toxins and their relation to hyperphosphorilation of cell proteins. Chem Biol Interact. 1992; 81:181-196.
Duy TN, Lam PKS, Shaw GR, Connell DW. Toxicology and risk assessment of freshwater cyanobacterial (blue-green algal) toxins in water. Reviews Environ Contam Toxicol. 2000;163:113-186.
Boelsterli UA. Mechanistic toxicology: the molecular basis of how chemicals disrupt biological targets. Informa Healthcare USA, Inc., New York. 2009. pp.233.
Carmichael WW. The toxins of cyanobacteria. Scient American. 1994;270:11, 64-70.
Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Eur J Physiol. 2004;447:653–665.
Fischer WL, Altheimer S, Cattori V, Meier PJ, Dietrich DR, Hagenbuch B. Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin. Toxicol Appl Pharmacol. 2005;203:257-263.
Meier-Abt F, Mokrab Y, Mizuguchi K. Organic Anion Transporting Polypeptides of the OATP/SLCO Superfamily: Identification of New Members in Non-mammalian Species, Comparative Modeling and a Potential Transport Mode. J. Membrane Biol. 2005;208:213–227.
Obaidat A, Roth M, Hagenbuch B. The Expression and Function of Organic Anion Transporting Polypeptides in Normal Tissues and in Cancer. Annu Rev Pharmacol Toxicol. 2012;52:135-151.
Leuthold S, Hagenbuch B, Mohebbi N, Wagner CA, Meier PJ, Stieger B. Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters. Am J Physiol Cell Physiol. 2009;296:C570–C582.
Hagenbuch B, Meier PJ. The superfamily of organic anion transporting polypeptides. Biochem Biophys Acta. 2003;1609:1–18.
Andrinolo D, Sedan D, Telese L, Aura C, Masera S, Giannuzzi L, Marra CA, Alaniz MJT. Hepatic recovery after damage produced by sub-chronic intoxication with the cyanotoxin microcystin LR. Toxicon. 2008;51:457-467.
Milutinović A, Sedmark B, Horvat-ŽnidarŠić I, Šuput D. Renal injuries induced by chronic intoxication with microcystins. Cell Mol Biol Lett. 2002;7: 139-141.
Milutinović A, Živin M, Zorc-Plesković R, Sedmark B. Šuput D. Nephrotoxic effects of chronic administration of microcystins-LR and –YR. Toxicon. 2003;42:281-288.
Robinson NA, Pace JG, Matson CF, Miura GA, Lawrence WB. Tissue distribution, excretion and hepatic biotransformation of microcystin-LR in mice. J Pharmacol Exp Therap. 1990;256:176-182.
Khan SA, Ghosh S, Wickstrom M, Miller LA, Hess R, Haschek WM, Beasley VR. Comparative Pathology of Microcystin-LR in Cultured Hepatocytes, Fibroblasts, and Renal Epithelial Cells. Nat Toxins. 1995;3:119-128.
McDermott CM, Nho CW, Howard W, Holtons B. The cyanobacterial toxin, Microcystin-LR, can induce apoptosis in a variety of cell types. Toxicon 1998;36(12):1981-1996.
Boaru DA, Dragoş N, Schrimer K. Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures cell lines: A comparative study. Toxicology. 2006;218:134-148.
Boyer JL, Hagenbuch B, Ananthanarayanan M, Suchy F, Stieger B, Meier PJ. Phylogenic and ontogenic expression of hepatocellular bile acid transport. PNAS. 1993;90:435–438.
Chong MWK, Gu K D, Lam PKS, Yang M, Fong WF. Study on the cytotoxicity of microcystin-LR on cultured cells. Chemosph. 2000;41:143-147.
Nong Q, Komatsu M, Izumo K, Indo H, Xu B, Aoyama K, Majima H, Horiuchi M, Morimoto K, Takeuchi T. Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxocity. Free Rad Res. 2007;41(12):1396-1337.
Žegura B, Sedmak B, Filipič M. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon. 2003;41:41-48.
Žegura B, Zajc I, Lah T, Filipič M. Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon. 2008;51:615-623.
Kullak-Ublick GA, Beuers U, Paumgartner G. Molecular and Functional Characterization of Bile Acid Transport in Human Hepatoblastoma HepG2 Cells. Hepatology. 1996;23:1053-1060.
Dias E, Pereira P, Batoréu MCC, Jordan P, Silva MJ. Cytotoxic and genotoxic effects of microcystins in mammalian cell lines. In: Moestrup, Ø. et al. (eds), Proceedings of the 12th International Conference on Harmful Algae, 4-8 September 2006, Copenhagen, Denmark. Paris: ISSHA and IOC- UNESCO; 2008.
Dias E, Andrade M, Alverca E, Pereira P. Batoréu MCC, Jordan P, Silva MJ. Comparative study of the cytotoxic effect of microcistin-LR and purified extracts from Microcystis aeruginosa on a kidney cell line. Toxicon. 2009;53:487-495.
Dias E, Matos P, Pereira P, Batoréu MCC, Silva MJ, Jordan P. Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney derived cell line Vero-E6. Toxicology in Vitro. 2010;24:1689-1695.
Alverca E, Andrade M, Dias E, Sam Bento F, Batoréu MCC, Jordan P, Silva MJ, Pereira P. Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosaextract on a kidney cell line. Toxicon. 2009;54:283-294.
Menezes C, Alverca A, Dias E, Sam Bento F, Pereira P. Involvement of endoplasmic reticulum and autophagy in microcystin-LR toxicity in Vero-E6 and HepG2 cell lines . Toxicology in vitro. 2013;27:138–148.
Hooser S. Fulminant Hepatocyte Apoptosis In Vivo Following Microcystin-LR Administration to Rats. Toxicol Pathol. 2000;28(5):726-733.
Mankiewicz J, Tarczynska M, Fladmark KE, Doskeland SO, Walter Z, Zalewski M. Apoptotic effect of cyanobacterial extract on rat hepatocytes and human lymphocytes. Environ Toxicol. 2001;16:225-233.
Li L, Xie P, Chen J. Biochemical and ultrastructural changes of the liver and kidney of the phytoplanktivorours silver carp feeding naturally on toxic Microcystis blooms in Taihu Lake, China. Toxicon. 2007;49:1042-1053.
MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS. 1990;264(2):187-192.
Toivola DM, Goldman RD, Garrod D, Ericksson JE. Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments. J Cell Sci. 1997;110:23-33.
Batista T, Sousa G, Suput JS, Rahmani R, Šuput D. Microcystin-LR causes the colapse of actin filaments in primary human hepatocytes. Aquatic Toxic. 2003;65:85-91.
Qin W, Xu X, Zhang X, Wang Y, Meng X, Miao A, Yang L. Endoplasmic reticulum stress in murine liver and kidney exposed to microcystin-LR. Toxicon. 2010;56:1334–1341.
Ding W, Shen H, Ong C. Microcystic Cyanobacteria Causes Mitochondrial Membrane Potential Alteration and Reactive Oxygen Species Formation in Primary Cultured Rat Hepatocytes. Environ Health Persp. 1998;106(7):409-413.
Ding W, Shen H, Ong C. Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology. 2000;2:547-555.
Ding W, Shen H, Ong C. Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes. Bioch Bioph Res Communic. 2002;291:321-331.
Weng D, Lu Y, Wei Y, Liu Y, Shen P. The role of ROS in microcystin-LR-induced hepatocytes apoptosis and liver injury in mice. Toxicology. 2007;232:15-23.
Žegura B, Lah T, Filipič M. The role of reactive oxygen species in microcystin-LR induced DNA damage. Toxicology. 2004;200:59-68.
IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Ingested Nitrate and Nitrite, and Cyanobacterial Peptide Toxins. Lyon, France. 2010;94:329-412.
Ueno Y, Nagata S, Tsutsumi T, Hasegawa A, Watanabe MF, Park HD, Chen GC, Chen G, Yu, SZ. Detection of microcystins, a blue-green algal hepatotoxins, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis. 1996;17:1317-1321.
Yu SZ. Primary prevention of hepatocellular carcinoma. J Gastroenterol Hepatol. 1995;10:674-82.
Zhou L, Yu H, Chen K. Relationship between microcystin in drinking water and colorectal cancer. Biomed Environ Sci. 2002;15:166-71.
Žegura B, Štraser A, Filipič M. Genotoxicity and potential carcinogenicity of cyanobacterial toxins – a review. Mut Res. 2011;727:16–41.
Collins AR. The comet assay for DNA damage and repair. Principles, applications and limitations, Mol Biotechnol. 2004;26:249–261.
Ding WX, Shen HM, Zhu HG, Lee BL, Ong CN. Genotoxicity of microcystic cyanobacterial extract of a water source in China. Mutat Res. 1999;442:69-77.
Žegura B, Gajski G, Štraser A, Garaj-Vrhovac V, Filipič M. Microcystin-LR induced DNA damage in human peripheral blood lymphocytes. Mut Res. 2011;726:116–122.
Bouaïcha N, Maatouk I, Plessis MJ, Périn F. Genotoxic potential of microcystin-LR and nodularin in vitroin primary cultured rat hepatocytes and in vivorat liver. Environ Toxicol. 2005;20:341-347.
Lankoff A, Krzowski L, Glab J, Banasik A, Lisowska H, Kuszwski T, Gozdz S, Wojcik A. DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR. Mut Res . 2004;559:131-142.
Zhan L, Sakamoto H, Sakuraba M, Wu S, Zhang S, Suzuki T, Hayashi M, Honma M. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells. Mut Res. 2004;557:1-6.
Lankoff A, Bialczyki J, Dziga D, Carmichael WW, Lisowska H, Wojcik A. Inhibition of nucleotide excision repair by microcystin-LR in CHO-K1 cells. Toxicon 2006;48: 957-965.
Fessard V, Le Hegarat L, Mourot A. Comparison of the genotoxic results obtained from the in vitro cytokinesis-block micronucleous assay with various toxins inhibitors of protein phosphatases: okadaic acid, nodularin and microcystin-LR. In: Proceedings of Sixth International Conference on Toxic Cyanobacteria, 21-27 June 2004, Bergen, Norway. 2004;68-69.
Abramsson-Zetterberg L, Sundh UB, Mattsson R. Cyanobacterial extracts amd microcystin-LR are inactive in the micronucleous assay in vivo and in vitro. Mutat Res. 2010;699:5-10.
Lankoff A, Bialczyki J, Dziga D, Carmichael WW, Gradzka I, Lisowska H, Kuszewski T, Gozdz S, Piorun I, Wojcik A. The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor. Mutagenesis. 2006;21:83-90.
Dias E, Louro H, Santos T, Antunes S, Pereira P, Silva MJ. Genotoxicity of Microcystin-LR in distinct biological models: cell lines, mouse blood cells and human lymphocytes. Environmental and molecular mutagenesis, submitted for publication.
Kirsch-Volders M, Vanhauwaert A, De Boeck M, Decordier I. Importance of detecting numerical versus structural chromosome aberrations. Mut Res. 2002;504:137-148.
Iarmarcovai G, Botta A, Orsière T. Number of centromeric signals in micronuclei and mechansims of aneuploidy. Toxicol Lett. 2006;166:1-10.
Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, Suganuma M, Kohyama K, Ishikawa T, Carmichael WW, Fujiki H. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J Cancer Res Clin Oncol. 1992;118:420–424.
Falconer IR. Tumour promotion and liver injury caused by oral consumption of cyanobacteria. Environ Toxicol Water Qual. 1991;6:177–184.
Gehringer MM. Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response. FEBS Lett. 2004;557:1–8.
Junttila MR, Li SP, Westermarck J.Phosphatase-mediated crosstalk between MAPK signalling pathways in the regulation of cell survival. FASEB J. 2008;22:954–965.
McKay MM, Morrison DK. Integrating signals from RTKs to ERK/MAPK. Oncogene 2007;26:3113-3121.
Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs. Oncogene. 2007;26:3100-3112.
Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 2000;351:289–305.
Li H, Xie P, Li G, Hao L, Xiong Q. In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins. Toxicon. 2009;53:169–175.
Zhu Y, Zhong X, Zheng S, Ge Z, Du Q, Zhang S. Transformation of immortalized colorectal crypt cells by microcystin involving constitutive activation of Akt and MAPK cascade. Carcinogenesis. 2005;26:1207–1214.
Thompson WL, Allen MB, Bostian KA. The effects of microcystin on monolayers of primary rat hepatocytes. In: Gopalakrisnakonef, P., Tan, C.K. (Eds.), Progress in venom and toxin research. National University of Singapore, Singapore, 1987;725–731.
Grabow W, Randt W, Prozesky O, Scott W. Microcystis aeruginosa toxin: cell culture toxicity, hemolysis and mutagenicity assays. Appl Environ Microbiol. 1982;43(6):1425–1433.
Menezes C. Comparative study of the cytotoxic effects of Microcystin-LR in mammalian cell lines. Universidade de Lisboa, 2009. Available from: http://repositorio.ul.pt/handle/10451/1488
Wickstrom ML, Khan SA, Haschek WM, Wyman JF, Eriksson JE, Schaeffer DJ, Beasle VR. Alterations in Microtubules, Intermediate Filaments and Microfilaments Induced by Microcystin-LR in Cultured Cells. Toxicol Pathol 1995;23:326-337.
Khan, S.A,. Wickstrom, M.L, Haschek, W.M., Schaeffer, D.J., Ghosh, S., Beasley, V.R. 1996.Microcystin-LR and kinetics of cytoskeletal reorganization in hepatocytes, kidney cells and fibroblasts. Nat. Toxins 4: 206-214.
Raju S, Kavimani S, Uma MRV, Sriramulu RK. Nephrotoxicants and Nephrotoxicity Testing: An Outline Of In Vitro Alternatives. J Pharm Sci Res. 2011;3:1110-1116.
Prieto P. Barriers, nephrotoxicology and chronic testing in vitro. ATLA. 2002;30:101-105.
Hawksworth GM, Bach PH, Nagelkerke JF, Dekant W, Diezi JE, Harpur E, Lock EA, MacDonald C, Morin PP, Pfaller W, Rutten AAJJL, Ryan MP, Toutain HJ, Trevisan A. Nephrotoxicity testing in vitro. The report and recommendations of ECVAM workshop 10. ATLA 1995;23:713-727.
Matskevich AA, Jung JS, Schümann M, Cascallo M, Moelling K. Vero Cells as a Model to Study the Effects of Adenoviral Gene Delivery Vectors on the RNAi System in Context of Viral Infection. J Innate Immun. 2009;1:389–394.
Jos A, Repetto G, Rios JC, Hazen MJ, Molero ML, del Peso A, Salguero M, Fernández-Freire P, Pérez-Martín JM, Cameán A. Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints. Toxicology in Vitro. 2003;17:525–532.
Jirasripongpun K, Wongarethornkul T, Mulliganavin S. Risk Assessment of Triclosan Using Animal Cell Lines. Kasetsart J Nat Sci. 2008;42(2):353-359.
Romero D, Gómez-Zapata M, Luna A, García-Fernández AJ. Morphological characterization of renal cell lines (BGM and VERO) exposed to low doses of lead nitrate. Histol Histopathol, 2004;19:69-76.
Freire PF, Peropadre A, Pérez Martín JM, Herrero O, Hazen MJ. An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines. Toxicology in Vitro. 2009;23:1553–1558.
Kumar S, Kanwar S, Bansal V, Sehgal R. Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum. Biologicals. 2009;37(5):297–305.
Lindgren SW, Samuel JE, Schmitt CK, O'Brien AD. The specific activities of Shiga-Like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia colidiffer when measured by Vero cell cytotoxicity but not by mouse lethality. Infect Immun. 1994;62(2):623-631.
Froscio SM, Fanok S, Humpage AR. Cytotoxicity Screening for the Cyanobacterial Toxin Cylindrospermopsin. J ToxicolEnviron Health, Part A: Current Issues. 2009;72(5):345-349.
Froscio SM, Cannon E, Lau HM, Humpage AR. Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells. Toxicon. 2009;54:862–868.
Ekwall B, Silano V, Paganuzzi-Stammati A, Zucco F. Toxicity Tests with Mammalian Cell Cultures. In: Bourdeau P. (ed.) Short-term Toxicity Tests for Non-genotoxic Effects. New York: John Wiley & Sons Ltd; 1990. p75-98.
Li T, Huang P, Liang J, Fu W, Guo Z, Xu L. Microcystin-LR (MCLR) Induces a Compensation of PP2A Activity Mediated by α4 Protein in HEK293 Cells. Int J Biol Sci. 2011;7:740-752.
Falconer IR. Cyanobacterial toxins present in Microcystis aeruginosa extracts – More than microcystins!. Toxicon. 2007;50:585–588.
Paulino S, Sam-Bento F, Churro C, Alverca E, Dias E, Valério E, Pereira P. The Estela Sousa e Silva Algal Culture Collection: a resource of biological and toxicological interest. Hydrobiologia. 2009;636:489–492.
Valério E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R. Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs. Microbiology. 2009;155:642–656.
Valério E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R. Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples. Environ Toxicol. 2010;25(3):251–260.
WHO. Guidelines for Drinking Water Quality, third ed. Incorporating the First and Second Addenda, vol. 1, Recommendations. World Health Organization, Geneva. 2008.