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Central European Journal of Immunology
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vol. 34

Review paper
The immunotoxic and nephrotoxic influence of cyanotoxins to vertebrates

Anna Rymuszka
Anna Sierosławska

Centr Eur J Immunol 2009; 34 (2): 129-136
Online publish date: 2009/05/20
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Toxic algae blooms are observed in many water bodies in Poland (e.g. Dobczycki Lake, Goczałkowicki Reservoir, Sulejówek Lake, Zemborzycki Reservoir, Baltic Sea) and constitute the worldwide and multifaceted research problem, having essential health implications for people and animals [1-3]. The cases of acute and chronic poisoning of aquatic organisms, including fish, farmed and domestic animals e.g. dogs, horses, cattle, birds living in the wild were reported. There are also data on intoxication of people being in contact with recreational or drinking water contaminated with cyanotoxins [4]. These substances have a various chemical structure (peptides, alkaloids) and the multidirectional mechanism of toxic action and are grouped into hepatotoxins, neurotoxins, dermatotoxin, cytotoxins, and toxins triggering other effects [5].
Long-term exposure to subclinical doses, which does not induce visible symptoms of poisoning can cause changes on the cellular level and can induce systemic dysfunctions. It is worthwhile noticing that sometimes organ changes are correlated with the decrease of the resistance to pathogens as the result of dysfunction of sensitive mechanisms of immunohomeostasis.

Nephrotoxic effects of cyanotoxins
Kidneys are the organ particularly exposed to the toxic action of xenobiotics. It is a consequence of its excretory functions and intensive blood flow through that tissue. The kidney is a complex organ where toxic changes can be observed, because many nephrotoxicants including drugs, chemicals and natural toxins and their metabolites are filtrated. The proximal tubular cells of the kidney very often can concentrate many nephrotoxins and hence are prone to the harmful effects of toxins.
There is a lot of data in the literature showing the influence of microcystins on the renal system of vertebrates (Table 1). Nephrotoxic effects of chronic administration relatively low doses (10 µg/kg i.p.) of microcystin-LR (MC–LR) and microcystin-YR (MC–YR) were studied by Milutinović et al. [20, 21]. Authors described many pathological changes in the kidneys of rats treated with MCs for 8 months. Degenerative changes in the kidneys were observed such as collapsed tufts of glomerular capillaries, the enlarged diameter of renal corpuscles, the widened Bowman’s space and proximal and distal convoluted tubules, thickened Bowman’s capsule in some renal corpuscles. Moreover, interstitial tissue was occasionally infiltrated by lymphocytes and appeared oedematous. Authors noted that the kidneys were far more affected than the liver. The cytoskeleton abnormalities and the DNA damage suggested, that the mechanisms underlying the chronic nephrotoxicity are similar at the cellular level to the mechanisms of the acute hepatoxicity of microcystins.
In the series of experiments performed by Nobre et al. [15, 18, 22, 23] it was shown that microcystin-LR can affect renal physiology. By using perfused rat kidney model authors showed an intense amount of proteinaceous material in urinary spaces following perfusion with MC-LR. Further studies confirmed that microcystin-LR promoted renal changes, such as altered vascular, glomerular and urinary parameters. MC-LR induced activation of phospholipase A2 (PLA2) and cyclooxygenase and this mechanism was similar to the mechanisms inducing hepatotoxic changes. Moreover, it was demonstrated that microcystin-LR stimulated macrophages to release the inflammatory mediators capable of promoting nephrotoxicity in the isolated perfused rat kidney. Authors examined the supernatant of macrophages stimulated in vivo by microcystin–LR and showed the presence of proinflammatory agents capable of provoking secretion of water and electrolytes (sodium, potassium and chloride). The observed collapsed filaments and other morphological changes support thesis that MCs could trigger apoptotic processes in the exposed kidney cells. That confirms the hypothesis that in vivo MCs induce cytoskeletal alterations and nuclear changes in different cells typical for appoptosis and/or necrosis [6, 13, 14, 26, 31, 35].
Rao et al. [19] suggests, that the observed cytotoxic effects leading to apoptosis were induced by generation of reactive oxygen species and caspase activation of cyanobacterial neurotoxins-anatoxin a in non-neuronal cells [19]. The results of this study showed that anatoxin-containing cell-free extracts from Anabena flos aquae and purified anatoxin-a induced concentration dependent cytotoxicity and apoptosis in African green monkey kidney cells (Vero). The authors observed morphological changes typical for apoptosis as plasma membrane blebbing, cell shrinkage, condensed chromatin, nuclear fragmentation and formation of DNA-containing apoptotic bodies. Several comparative studies have shown that microcystins develop the same cytotoxic response [8-10].
It was revealed by the immunostaining that the injected conjugates can accumulate in the kidneys [36]. It might be thus speculated that in the conditions of chronic exposure to MC-LR accumulation of its metabolites in the kidneys and changes in their physiology may occure. Kotak et al. [11] indicated that kidney tubular epithelial cells in fish were affected after acute exposure by interperitoneal injection of MC-LR 400 µg/kg and 1000 µg/kg. Similarly, Radbergh et al. [7] have shown degenerative changes in the tubular epithelial cells, glomeruli and interstitial tissue in kidneys of carp intraperitoneally exposed to MC-LR with the LD50 ranging from 80 to between 300 and 550 µg/kg. The studies also indicated that fish can tolerate higher doses of the toxin and have a longer survival period compared to mice. Probably the uptake of microcystin to the kidney may be dependent on body temperature.
Fisher and Dietrich [16] found microcystin-induced alterations in kidney tissues of carp when Microcystis aeruginosa (PCC 7806) amounting to an equivalent of 400 µg MC-LR/kg bw were directly administered to the fish stomach. In the kidney degenerative changes were observed in the renal proximal tubules, a segment known for its high capacity of active protein and peptide reabsorption. Moreover, the studies on the mechanism of cell toxicity showed that cyanotoxins induce oxidative stress in tissues of vertebrates and the potential alterations of the antioxidant status [25, 27, 30, 34].

Immunotoxic effects of cyanotoxins
Many substances present in the aquatic environment at relatively low concentrations demonstrate toxic action on the immune cells and organs of fish and higher vertebrates. Immune system, together with other systems e.g. nervous and endocrine, takes part in regulating homeostasis, so cyanotoxins, which have multidirectional nature of the action can also induce directly or indirectly dysfunctions of the immune system. The immunotoxic effects of cyanotoxins are summarised in Table 2. Immunosuppression was also confirmed in our study, which determined the influence of microcystin-LR and anatoxin-a to the basic functions of the fish immune cells [56, 57, 60].
The obtained findings showed the inhibition of the viability of lymphocytes and phagocytes isolated from rainbow trout by the toxin in the time and concentration dependent manner. Microcystin-LR suppressed the examined functions of immune cells (metabolic activity of phagocytes, proliferative response of lymphocytes) more distinctively compared to anatoxin-a (in press). Moreover, we noted that phagocytes are more sensitive to microcystin-LR than lymphocytes. It is interesting, that extracts containing the cyanotoxins are more immunotoxic, than the pure form toxins (unpublished).
Moreover, other authors describe the decrease of the total number of white blood cells, including T lymphocytes (particularly cytotoxic T cell), B cells, mielocytes and the lowered value of the phagocytic index after the exposure to microcystins [37, 47, 59].
The mechanism of toxic action of microcystins is the blockade of the activity of protein phosphatases serine (PP1) and threonine (PP2) what leads to hyperphosphorylation of plasmic and cellular cytoskeleton proteins. The disorders of the intracellular homeostasis can result in the uncontrolled proliferation and as a consequence induce carcinogenesis [19, 41, 44, 50, 52, 54, 55, 59, 61].
The activation of the phagocytic cells is connected with a sequence of changes in their cytoskeleton. Our studies showed that phagocytes may be the target cells for toxic effects of the microcystin-LR in the fish immune system [57, 60]. The toxin at the concentrations environmentally relevant caused the increased production of reactive forms of oxygen in phagocytes and modulation of the phagocytosis. The disorders of the metabolic activity of these cells can result from the direct effects of the toxin on their cytoskeleton which influence the activation and process of phagocytosis.
Our studies indicated that microcystin-LR is more suppressive to B lymphocytes, than to T cells [56]. Differences in the response of the two lymphocyte populations to the cyanotoxin probably result from the action of cytokines – essential regulatory proteins of the immune system, as well as nervous and endocrine systems. This hypothesis is confirmed by studies carried out on mammalian immune cells. These studies showed that microcystin-LR influenced the production of cytokines: interleukin-2 (IL-2) and interleukin-6 (IL-6) responsible for lymphocyte functioning [45]. Moreover, the influence of this toxin on the expression of cytokines such as: IL-1b, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-g, as well as on the nitric oxide synthase activity in phagocytes was observed [39, 42, 48, 49, 51, 52, 58].
In summary, our research and the observations of other authors suggested that cyanotoxins may induce nephrotoxic and immunotoxic effects and changes in physiology of vertebrates.

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Copyright: © 2009 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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