Cylindrospermopsin: Water-linked potential threat to human health in Europe

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 651–660

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Cylindrospermopsin: Water-linked potential threat to human health in Europe b,c ´ Barbara Poniedziałek a,∗ , Piotr Rzymski a , Mikołaj Kokocinski a b c

´ Poland Department of Biology and Environmental Protection, Poznan University of Medical Sciences, Poznan, ´ Poland Department of Hydrobiology, Adam Mickiewicz University, Poznan, Collegium Polonicum, Faculty of Biology, Adam Mickiewicz University, Słubice, Poland

a r t i c l e

i n f o

a b s t r a c t

Article history:

Cylindrospermopsin (CYN) is a secondary metabolite produced by several cyanobacteria

Received 7 April 2012

species. Its potential effect on human health includes liver, kidneys, lungs, spleen and intes-

Received in revised form

tine injuries. CYN can be cyto- and genotoxic to a variety of cell types. Occurrence and

2 August 2012

expansion of species able to synthesize CYN in European water bodies has been recently

Accepted 22 August 2012

reported and raised awareness of potential harm to human health. Therefore, surface water

Available online 30 August 2012

of different human use should be monitored for the presence of toxic species of blue-green

Keywords:

potential effects of the toxin on human health according to the current state of knowledge.

algae. This paper aims to describe the distribution of CYN producers in Europe and the Cylindrospermopsin

© 2012 Elsevier B.V. All rights reserved.

Toxicity Cyanobacteria Mechanism of action

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution of potential CYN producers in Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioaccumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential impact of CYN on human’s health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Potential routes of exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Clinical cases of poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Immunotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Cytotoxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: Department of Biology and Environmental Protection, Poznan University of Medical Sciences, ul. Długa 1/2, ´ Poland. Tel.: +48 61 854 91 78; fax: +48 61 854 91 78. 61-848 Poznan, E-mail address: [email protected] (B. Poniedziałek). 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.08.005

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5.

1.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 651–660

4.9. Dermatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10. Cancerogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11. Fetal toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

Blue-green algae (cyanobacteria) are a group of prokaryotic, autotrophic microorganisms that contain the photosynthetic pigments (chlorophyll and phycocyanin). So far over 2500 species have been identified, mainly related to ecosystems of surface waters, both marine and freshwater (Cavalier-Smith, 2002; Oren, 2011). Several cyanobacterial species are able to synthesize secondary metabolites highly toxic to mammals, including humans (Mazur-Marzec, 2006; Apeldoorn et al., 2007). Depending on the target of action cyanotoxins can be classified as dermato-, cyto-, neuro- and hepatotoxins (Apeldoorn et al., 2007; Pearson et al., 2010). Bioaccumulation of these toxic compounds in aquatic animals has also been shown (Ibelings and Chorus, 2007). Since cyanobacteria are observed to be spreading widely across the world due to climate changes and eutrophication (Vasconcelos, 2006; Paerl and Huisman, 2009), it has been proposed that water reservoirs and aquatic livestock should be subject to regular quality control monitoring (Chorus and Bartman, 1999). Cylindrospermopsin (CYN) is a newly emerging toxin originally identified in tropical cyanobacteria Cylindrospermopsis raciborskii (Mazur-Marzec, 2006; Bownik, 2010). CYN (CAS number 143545-90-8) is polyketide-derived alkaloid with a central functional guanidino moiety combined with hydroxymethyluracil attached to its tricyclic carbon skeleton (Fig. 1). First identification and isolation dates back to 1992 (Ohtani et al., 1992). Structural variants of 7-epi-CYN (an epimer at the hydroxyl bridge) and 7-deoxy-CYN (lacking the hydroxyl group) has been latterly reported (Banker et al., 2001; Seifert et al., 2007). CYN has a relatively low molecular weight of 415 Da and is highly soluble in the water (Sivonen and Jones, 1999). It has been shown to be very stable in the visible as well as UV light, and over wide range of pH and temperature

Fig. 1 – Chemical structure of cylindrospermopsin and analogues.

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changes. Repeated boiling of water does not destroy nor inactivate the toxin (Chiswell et al., 1999; Wörmer et al., 2010). Degradation of CYN in surface waters is promoted by oxic conditions and inhibited in anoxic environment (Klitzke and Fastner, 2012). CYN is highly mobile during sediment passage and its sorption depends on organic carbon availability (Klitzke et al., 2010). For most known cyanotoxins intracellular concentration tends to be higher than extracellular. However, Rücker et al. (2007) noted that concentration of CYN dissolved in the water can constitute as much as 90% of total CYN available. Therefore, the risk of human exposures and intensity of health effects can be higher than for any other cyanotoxin. During massive proliferation of CYN-producers in surface waters its concentration in water can exceed 500 ␮g L−1 (Shaw et al., 1999). Humpage and Falconer (2003) proposed 1 ␮g L−1 as a guideline safety value of CYN in drinking water although in France the maximum allowed CYN concentration cannot exceed 0.3 ␮g L−1 (AFSSA, 2006). It seems reasonable to develop appropriate regulations for CYN also in other European countries. Recently several publications reported the occurrence of C. raciborskii and other potential CYN-producers in the European water bodies. Wide spectrum of potential impact of CYN on human health has been described. Observations have been made in animal model experiments as well as on human cell lines. This paper aims to describe the distribution of cyanobacteria species producing CYN identified in surface waters of Europe as well as potential impact of this toxin on human health according to the current state of knowledge.

2. Distribution of potential CYN producers in Europe Eleven freshwater cyanobacteria species have been identified to produce the CYN: C. raciborskii, Aphanizomenon ovalisporum, Aphanizomenon flos-aquae, Aphanizomenon gracile, Aphanizomenon klebahnii, Umezakia natans, Raphidiopsis curvata, Anabaena bergii, Anabaena planctonica, Anabaena lapponica and Lyngbya wollei (Bláhová et al., 2009; Pearson et al., 2010). Among them C. raciborskii, A. ovalisporum, A. gracile, A. flos-aque, A. lapponica, A. bergii, A. planctonica, A. klebahnii (belonging to Nostocales order) and L. wollei (belonging to Oscillatoriales order) have been identified in European surface waters. These species can form blooms induced by increased levels of nitrogen and phosphorus, elevated temperature and light, leading to a mass reproduction of specific cyanobacterium species for several days. So called “harmful algae blooms” (HAB) usually occur in the summer and autumn seasons (Reynolds ´ and Walsby, 1975; Burchardt and Pawlik-Skowronska, 2005). Expansion of C. raciborskii (originally considered to be a pantropical species) into the temperate regions has been

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observed during last decades. In Europe this species was identified for the first time in Lake Kastoria (Greece) in 1938 (Skuja, 1938), then in 1970 in Lake Balaton, Hungary (Padisák, 1997), in 1979 in Borycki Reservoir, Slovakia (Horecká and Komárek, 1979) and subsequently in other European countries such as Austria in 1993 (Dokulil and Mayer, 1996), Spain and France in 1994 (Romo and Miracle, 1994; Couté et al., 1997) and Portugal in 2002 (Saker et al., 2002). However, C. raciborskii is widely occurring cyanobacterium in Europe, so far CYN-producing strain of this species has not been identified here (Neilan et al., 2003; Haande et al., 2008; Mankiewicz-Boczek et al., 2012). The reason why European C. raciborskii strains do not synthesize CYN could be associated with the absence of cyrJ gene (Mihali et al., 2008). However, it cannot be entirely excluded that new CYN-producing strains of C. raciborskii will appear in European countries. Bonilla et al. (2011) suggest future predominance of this species under predicted climate-change scenarios. However, CYN-producing strains of C. raciborskii have not been observed in Europe, CYN was detected in many European countries indicating other cyanobacteria to be a potential CYN producers, including A. flos-aquae in Germany (Preussel et al., 2006), A. gracile in Germany (Rücker et al., 2007; Wiedner ´ et al., 2009), A. lapponica et al., 2008) and Poland (Kokocinski in Finland (Spoof et al., 2006), A. flos-aquae and A. planctonica in France (Brient et al., 2009), A. klebahnii in Czech Republic (Bláhová et al., 2009) and A. ovalisporum in Italy (Messineo et al., 2010). CYN is being identified in Europe since 2000 when its occurrence (0.2 ␮g L−1 ) was found for the first time in German lakes (Fastner et al., 2003). An extensive survey was conducted by Messineo et al. (2009) in which authors analyzed concentration of different cyanotoxins in 28 Italian lakes used for recreation and drinking water abstraction. CYN concentration varied from 0.3 to 123 ␮g L−1 . Similar study conducted on 21 lakes in Germany revealed maximal detected CYN concentration of 12.1 ␮g L−1 (Rücker et al., 2007). The frequency of CYN occurrence in northeast Germany is similar to that in Australia and North America and equal to that of microcystin (Fastner, 2007). CYN level in surface water was also determined in Czech Republic (0.4–4.4 ␮g L−1 ), France (from trace quantities to 1.95 ␮g L−1 ) and Poland (0.16–1.8 ␮g L−1 ) (Bláhová et al., ´ 2009; Brient et al., 2009; Kokocinski et al., 2009). It should be highlighted that the development of CYN-producing species in surface water of any human use can pose a potential public health threat. Please note that measured levels of CYN exceeds WHO 1 ␮g L−1 guideline safety of Humpage and Falconer (2003) in all European countries the toxin was identified. Therefore, CYN shall be a subject of routine and regular monitoring in countries where cyanobacterial blooms are frequently being reported.

3.

Bioaccumulation

Similar to some other cyanotoxins (e.g. microcystins and nodularins), CYN can also accumulate in tissues of aquatic organisms, including invertebrates and vertebrates (Saker et al., 2004; Ibelings and Chorus, 2007; Berry et al., 2012). Moreover, CYN can bioaccumulate in organisms even if they are exposed to trace quantities of the toxin.

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Proposed general order of bioaccumulation capacity is gastropods > bivalves > crustaceans > amphibians > fish (Kinnear, 2010). Recently Niedzwiadek et al. (2012) conducted investigation of CYN (and other cyanotoxins) in food products (e.g. shrimps and farmed fish) sold in Canada and found absence of the toxin. It has been found that some of bluegreen algae based products (e.g. Arthospira platensis commonly used for diet supplementary) can contain cyanobacterial toxins (e.g. microcystins, anatoxins) when cells are harvested from natural lakes in which other species can also be identified (Rellán et al., 2009; Hutadilok-Towatana et al., 2010). So far these products has been found to be CYN-free (Liu and Scott, 2011). However, no studies found CYN in livestock used for consumption, contamination of food products with this toxin cannot be entirely ruled out and requires thorough research.

Potential impact of CYN on human’s 4. health 4.1.

Mechanism of action

Uracil moiety and the hydroxyl group are believed to be crucial for CYN toxicity (Banker et al., 2001). How CYN enters intracellular environment is not yet understood. Molecular weight and hydrophilic properties suggests that toxin unlikely readily cross lipid bilayer. Chong et al. (2002) suggested that the bile acid transport system could partially aid the transport of CYN into primary hepatocytes. On the other side, Froscio et al. (2009a) demonstrated that CYN is not actively transported into the cells (using Vero cell line model). Sulfate group was shown not to be responsible for cellular uptake (Runnegar et al., 2002). It is unknown whether uracil moiety has any importance in this process. CYN has a potential to interfere with several metabolic pathways. Rapid toxicity seems to be mediated by cytochrome P450 (Runnegar et al., 2002; Norris et al., 2002; Froscio et al., 2003). It has been shown that CYP1A1 and CYP1A2 enzymes are involved in CYN metabolic activation. However, possible involvement of other CYP isoforms cannot be excluded ˇ ˇ et al., 2011; Zegura et al., 2011). The longer-term (Straser toxicity of CYN seems to be due to irreversible inhibition of protein synthesis and inhibition of glutathione synthesis (GSH), which potentially can lead to increase in oxidative stress (Runnegar et al., 1995; Froscio et al., 2009a). Unlike microcystin, CYN does not inhibit phosphatases 1, 2A or 3 (Runnegar et al., 1994, 1995; Chong et al., 2002). Ingested CYN is transferred from the gastrointestinal tract to the systematic circulation, and is transported mainly to liver, however, as has been shown in rodent experiments, the other organs (kindey, lungs, thymus, spleen, adrenal glands, heart) can also be a target of CYN (Hawkins et al., 1985; Terao et al., 1994; Falconer et al., 1999; Humpage et al., 2000a,b; Norris et al., 2001; Oliveira et al., 2012). Limited knowledge exists on potential neuro-, immuno-, dermato-, cyto-, and fetal toxicity in human. Recently valuable studies involving human cell lines were conducted and revealed cyto- and genotoxic effects of CYN.

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4.2.

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Potential routes of exposure

Based on confirmed clinical cases (described later), bioaccumulation tendency and knowledge gained from other cyanotoxins studies (e.g. microcystin, lyngbyatoxin) routes of potential human exposure to CYN can include:

1. 2. 3. 4.

Drinking contaminated water Consuming contaminated food Bathing or showering in contaminated water Recreational water activities (swimming, wading, boating, water skiing, etc.) during cyanobacterial bloom.

4.3.

Clinical cases of poisoning

So far, only two epidemic cases have been retrospectively confirmed to be caused by CYN. In 1979, a major outbreak of hepatoenteritis occurred in Palm Island, Australia. Altogether, 140 children and 10 adults required hospitalization. The recognized symptoms included malaise, anorexia, vomiting, headache, bloody diarrhea, dehydration and painful hepatomegaly. A severe acute kidney disease occurred, resulting in electrolyte loss, ketonuria, glycosuria and hematuria. Another finding was liver failure, with elevated serum liver enzymes. All of the patients recovered with supportive treatment of dehydration and hypovolemic shock. The epidemic outbreak was directly linked to the potable water reservoir (Solomon Dam) in which C. raciborskii bloomed at that time. The bloom was treated with 1 ppm of copper sulfate – a popular algaecide leading to cyanobacterial cell lysis. This resulted in a massive release of CYN from the cells into the water although actual toxin concentration to which people were exposed in drinking water remains unknown (chemical structure of CYN was not known until 1992). Once ingested, copper sulfate has not been shown to cause the above symptoms, since concentration of copper used to erase the algae from the water was in the safe range (Byth, 1980; Bourke et al., 1983; Griffiths and Saker, 2003). All affected individuals had consumed water from the Solomon Dam reservoir. Based on this particular case it might be assumed that the risk of CYN exposure can be greater in children (due to the higher consumption of water per unit of body weight or possibly higher biologically susceptible to toxin than adults). Second case of CYN toxicity in humans is related to Caruaru (Brazil) tragedy in 1996 at local dialysis center. No use of reverse osmosis in filtration system resulted in the presence of microcystins and CYN in water supply followed by death of over 50 exposed patients. Detected content of CYN in carbon, ion-exchange resin, and sand from the in-house filters at dialysis clinic was in range of 0.02–19.7 ␮g g−1 . To compare, microcystin content valued from 0.5 ␮g g−1 to 3.4 ␮g g−1 . Unfortunately water treatment personnel were not performing any phytoplankton counts and investigation as well as no cyanotoxins level was determined during the time the tragedy occurred (Carmichael et al., 2001). A pattern of disease (which is now referred to Caruaru syndrome) was manifested by painful, extreme hepatomegaly, jaundice and a bleeding diathesis manifested by ecchymosis, epistaxis, and methrorrhagia; elevated transaminases, variable hyperbilirubinemia,

prolonged prothrombin time, and severe hypertrigliceridemia. Histologically disruption of liver plates, liver cell deformity, necrosis, apoptosis, cholestasis, cytoplasmic vacuolization, mixed leukocyte infiltration and multinucleated hepatocytes was observed by light microscopy and intracellular edema, mitochondrial changes, rough and smooth endoplasmic reticulum injuries, lipid vacuoles and residual bodies observed upon electron microscopy (Jochimsen et al., 1998; Pouria et al., 1998). It should be noted that observed symptoms are most probably combined effect of microcystin and CYN toxicity. In reality, the number of human poisonings involving CYN can be greater as many cases may have not been identified because of complicated CYN detection procedures or lack of awareness and knowledge of cyanobacterial toxins. In addition, numerous cases of animals’ poisoning (e.g. cattle) after drinking water from dam dominated by C. raciborskii (actual concentration of CYN remains unknown), including lethal cases, have been recorded. Staggering and weakness before death was observed in animals. Necropsy revealed hyperemic mesenteries, pale and swollen liver. Histopathologic image of calf carcass livers showed hepatic degeneration and necrosis and deposits of fibrous tissue throughout the liver (Thomas et al., 1998; Saker et al., 1999; Briand et al., 2003).

4.4.

Hepatotoxicity

Ingestion of CYN can lead to gastroenteritis through injury to the gut lining and hepatitis from injury to liver cells. Four phases of pathomorphological changes induced by CYN in hepatocytes can be divided. The initial phase is represented by reduction of nucleoi size and ribosomes detachment from membranes as well as their accumulation into the cytoplasm. The second phase can be characterized by marked proliferation of agranular membranes due to lipid peroxidation induced by decrease of cytochrome P450 amount. The third phase is manifested by an accumulation of fat droplets in the central portion of hepatic lobules (probably induced by free radicals). In the last phase severe liver necrosis can be observed (Duy et al., 2000; Apeldoorn et al., 2007). Bernard et al. (2003) conducted toxicological comparison between cell extracts of different C. raciborskii strains in IOPS OF1 (Swiss Albino) mice bioassay and found that although Hungarian strain (ACT 9502) did not pose any toxicity after 48 h of exposure, intraperitoneal injection of French strain (PMC 98.14) extract (1000 mg kg−1 body weight) caused liver damage with multifocal necrosis characterized by small areas of hepatocellular necrosis, combined with disorganization of the parenchyma and congestion of the inner sinusoid. Loss of intracellular glycogen stores in hepatocytes was also suggested due to observed darker cytoplasm than in control samples. The toxicity of French strain was considerably lower than that of an extract from Australian strain of C. raciborskii. In other studies intraperitoneal injection of sonicated C. raciborskii cells or pure CYN led to centrilobular damage of hepatocytes (Hawkins et al., 1985, 1997; Chernoff et al., 2011). Chernoff et al. (2011) observed liver injury manifested by increased alanine aminotransferase, aspartate amino transferase and sorbitol dehydrogenase in CD-1 (Swiss-Webster) mice after intraperitoneal injection of 50 ␮g CYN kg−1 administered daily for 4 days. In oral studies significant increase in

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liver weight was observed in ICR mice receiving 0.6 mg CYN L−1 (0.066 mg kg−1 bw) via drinking water for 3 weeks (Reisner et al., 2004). Similar effects were also reported in mice receiving aqueous extracts of C. raciborskii (43–135 mg kg−1 bw) via drinking water for 10 weeks (Humpage and Falconer, 2003). Recent study by Oliveira et al. (2012) has shown that CYN was detectable in the cytosol of hepatocytes of all treated Balb/c mice (70 ␮g CYN kg−1 bw, intratracheal injection) after 2 h of exposure (40 ng g−1 tissue). Significant higher values were recorded after 96 h of exposure (80 ng g−1 tissue). Clear hepatotoxicity of CYN has also been shown in two confirmed cases of human (Australian and Brazilian) and numerous animals’ poisoning as already described in ‘Clinical cases of poisoning’ subchapter.

4.5.

Neurotoxicity

The neurotoxicity of CYN is controversial. There are no studies involving human cell lines and no neurotoxicity was observed in two confirmed cases of human poisoning. However, it was found that a crude extract of C. raciborskii culture either bath-applied by microperfusion or locally delivered to the cell surface had a direct depolarizing or hyperpolarizing effect on neurons of two snail species – Helix pomatia and Lymnaea stagnalis (Kiss et al., 2002). Also alligators found in lake Griffin (Florida) during C. raciborskii bloom demonstrated depressed clinical responses, reduced nerve conduction velocities, axonal degeneration, and necrosis of specific loci in the midbrain (Schoeb et al., 2002). Zagatto et al. (2012) studied neurotoxic effect of intraperitoneally injected C. raciborskii extract of toxic strains (T2 and T3) in Swiss mice. Dosing with 50 mg kg−1 bw revealed typical symptoms of neurotoxicity such as tremor, ataxia, convulsions, and death by respiratory failure within 1–2 min. The same effects were also observed when mice were treated with boiled extract or filtered in AP20 membrane. Changes of extract pH (adjustment to 1.2 and 12) also did not decrease its toxicity. Also Saker et al. (2003) observed neurologic symptoms on Charles-River mice after intraperitoneal injection of C. raciborskii (1337–1572 mg kg−1 bw). Mice exhibited symptoms of piloerection, lethargy and difficulty in breathing leading to death within maximum 24 h after exposure. Treated mice did not consume any food or drink any water (Saker et al., 2003). On the other side it was also shown that some C. raciborskii strains can produce, beside CYN, other toxic compound – saxitoxin (STX) that is primarily neurotoxic (Soto-Liebe et al., 2010) although it was not detected in Saker et al. (2003) study. Further analyses in this area of study must be conducted and neurotoxicity action of CYN cannot be entirely ruled out.

4.6.

Genotoxicity

Several observations on different human cell lines were conducted and revealed genotoxic properties of CYN. It was recently reported that this toxin induces MNi (micronuclei) formation in human liver-derived HepG2 cells along with nuclear bud (NBUD) and nucleoplasmic bridge (NPB) formation, with the cytokinesis block micronucleus (CBMN) assay ˇ et al., 2011). after 24 h exposure to 0.05 and 0.5 ␮g ml−1 (Straser MNi formations induced by CYN have been also reported

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in differentiated HepaRG cells exposed to 15 ␮g ml−1 and undifferentiated HepaRG cells exposed 24 h to 30 ␮g ml−1 , the colon-derivated Caco-2 cells (differentiated and undifferentiated) exposed 24 h to 0.5–2 ␮g ml−1 (Bazin et al., 2010), lymphoblastoid cell line WIL2-NS exposed 48 h to 1–10 ␮g ml−1 (Humpage et al., 2000a,b), and peripheral blood lymphocytes ˇ exposed 4 h to 0.5 ␮g ml−1 and 24 h to >0.1 ␮g ml−1 (Zegura et al., 2011). These results indicate aneugenic and clastogenic activity of CYN that can lead to structural and numerical chromosome aberrations. Humpage et al. (2000a,b) suggested that CYN can also induce DNA strand breaks, what recently ˇ was demonstrated by Straser et al. (2011) in human HepG2 cell line after 12 h exposure to 0.5 ␮g ml−1 and after longer (24 h) treatment with lower CYN concentrations (0.01, 0.05 ˇ and 0.1 ␮g ml−1 ). Similar results were obtained by Zegura et al. (2011) in human peripheral blood lymphocytes after 4 h exposure to 0.5 ␮g ml−1 and 24 h exposure to lower CYN concentrations (0.05 and 0.1 ␮g ml−1 ). Moreover up-regulated expression of genes involved in the response to DNA damage (MDM2 and GADD45␣), apoptosis (BCL-2 and BAX) and oxidative stress (GPX1, SOD1, GSR, GCLC) were observed after 24 h treatment with 0.5 ␮g CYN ml−1 while no noticeable alterations occurred after 4 h of exposure. The same CYN concentration up-regulated expression of MDM2 and GADD45␣ genes in human HepG2 cell line after 12 h and 24 h of expoˇ sure (Straser et al., 2011). There are also several reports from studies in animal model. Shen et al. (2002) observed significant DNA strand breakage in hepatocytes of Balb/c mice 24 h after administrating single dose of 0.2 mg CYN kg−1 bw (intraperitoneal injection). CYN or its metabolites have been shown in vivo to bind to liver DNA in Quackenbush mice, forming adducts, 24–96 h after being treated with a single dose (intraperitoneal injection) of 1 ␮g CYN kg−1 (Shaw et al., 2000). Fessard and Bernard (2003) who studied effect of CYN (0.5 and 1 ␮g ml−1 ) on Chinese hamster ovary K1 cells in vitro did not observed any DNA damage after 24 h of treatment although strong inhibition of cell growth, cell blebbing and rounding occurred – morphological effects rather linked to cytoskeletal reorganization than apoptosis. CYN genotoxicity was also found by Chernoff et al. (2011). Daily administration (4 days) of 50 ␮g CYN kg−1 bw (intraperitoneal injection) in CD-1 (SwissWebster) altered the expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress. Above citied studies clearly confirm that CYN has a potential to enter and affect the variety of cell types.

4.7.

Immunotoxicity

Effect of CYN on immune response is not well studied so far. Terao et al. (1994) reported massive lymphocytes necrosis in the cortical layer of the thymus in ICR mice after intraperitoneal injection of 0.2 mg CYN kg−1 bw. Degeneration and necrosis of cortical lymphocytes as well as lymphophagocytosis in the lymphoid tissue of the spleen was observed in MF1 mice after exposure to 4.4–8.3 mg CYN kg−1 bw (Seawright et al., 1999). CYN induced lymphophagocytosis in the spleen of Quackenbush mice was also shown at dosing with the cellfree extract at 0.05 mg CYN kg−1 bw for 14 days. However,

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dosing with purified CYN did not cause any lymphophagocytosis (Shaw et al., 2000, 2001). Further studies are necessary to determine whether CYN can be classified as a immunotoxicant.

4.8.

Cytotoxicity

CYN has a possibility to enter a variety types of cells and cause toxic effects (Froscio et al., 2009b). Such attribute of this toxin has already been described in ‘Genotoxicity’ subchapter. Cytotoxicity to human CKO-K1 cells has been shown resulting in increased necrosis and decreased proliferation and mitotic indices in concentration (0.05–2 ␮g CYN ml−1 ) and time (3–21 h) dependent manner (Lankoff et al., 2007). In human HepG2 cells treated with 2.5 ␮g CYN ml−1 increase in the number of floating cells was observed (Neumann et al., 2007). Gutiérrez-Praena et al. (2012a) studied biochemical and pathological effects of CYN on human Caco-2 cell line and found ultrastructural alterations, which reveal lipid degeneration, mitochondrial damage and nucleolar segregation with altered nuclei – all noticeable at the lowest assayed concentration (1.25 ␮g CYN ml−1 ) after 24 h and 48 h of exposure. Simultaneously an increase in reactive oxygen species (ROS) production in concentration (0.625–2.5 ␮g CYN ml−1 ) dependent manner has also been shown. Increase of ROS production was also observed in endothelial HUVEC cell line by GutiérrezPraena et al. (2012b) only when exposed for 24 h to the lowest assayed concentration 0.375 5 ␮g CYN ml−1 . Higher concentrations (0.75–1.5 ␮g ml−1 ) decreased it to the level of the control group. It should be noted that sustained overproduction of ROS may result in oxidative stress which is associated with detrimental effects, namely alterations on the normal function of lipids, proteins or DNA as well as with aging, arteriosclerosis and cancer (Gate et al., 1999; Freitas et al., 2009). The highest CYN concentration assayed (40 ␮g ml−1 ) reduced Caco-2 cells viability by 90% (Gutiérrez-Praena et al., 2012a). Significant decrease of cell viability was observed in endothelial HUVEC cell line after 48 h exposure to 40 ␮g ml−1 of CYN. Observed morphological alterations after 24 exposure to 1.5 ␮g CYN ml−1 included nucleolar segregation with altered nuclei, degenerated Golgi apparatus, increases in the presence of granules and apoptosis. Longer time of incubation (48 h) resulted in greater cells injury (Gutiérrez-Praena et al., 2012b).

during swimming or bathing in water contaminated with CYN. There are no confirmed cases of CYN dematotoxicity in human although dermal effects were often observed after contact with cyanobacteria blooming water. However, it should be noted that several other secondary metabolites produced by some cyanobacteria (e.g. lyngbya-, aplysiaand debromoaplysiatoxin) have evident dermal effect in humans during a direct exposition (Rzymski and Poniedziałek, 2012). Dermatotoxicity of CYN-producers can be also a result of lipopolysaccharides (LPS) presence in cyanobacterial cell walls (Stewart et al., 2006b). Therefore, all CYN-producible cyanobacteria should be considered as potentially dermatotoxic to humans.

4.10.

International Agency for Research on Cancer (IARC) concluded in 2006 that there is no sufficient available data to resolve the question whether CYN can be involved in carcinogenesis processes. (Grosse et al., 2006). So far there is only one report of CYN-initiated tumor observed in mouse bioassay. Five tumors (among them two hepatic dysplatic foci and one frank HCC) were found in 53 Swiss mice treated for 30 weeks with C. raciborskii extract (2500 mg kg−1 bw) containing 13.8 mg of CYN, while none were found in the 27 controls. TPA was administered to some treatment groups with the aim of promoting the growth of initiated tumors. There was no experimental evidence for this occurring. Although the number of animals was too low to provide statistical significant evidence of cancerogenesis, the range of the calculated relative risk (RR = 6.2; 95% CI: 0.33–117) suggests that it would be imprudent to reject this possibility. However, the study design was limited due to short period of exposure, it certainly highlights the need for further investigation due to raised public health considerations (Falconer and Humpage, 2001). The other study, a follow-up review of medical records from the children poisoned from the Australian outbreak in 1979 (see ‘Clinical cases of poisoning’ subchapter) found an increased rate of gastrointestinal cancers in the period of 1982–1999 when compared to an unexposed similar population although no significance was found due to the low number of individuals in the exposed population (Falconer and Humpage, 2006).

4.11. 4.9.

Cancerogenesis

Fetal toxicity

Dermatotoxicity

CYN can also be dermatotoxic. Torokne et al. (2001) exposed intradermally albino Californian rabbits to 0.2 ml of lyophilized A. ovalisporum extract and observed erythema and oedema after 24 h although skin irritation was described as moderate. Topical application (abdominal skin) of 100 ␮g CYN ml−1 in Balb/c mice resulted in dried skin, yellow/brown crusts, desquamation and blood or serous fluid oozing from exposed skin. Lesions produced on skin were noted from the second induction day onwards. Ears of mice treated with 100 ␮g CYN ml−1 resulted in oedema, thickening, and inflammatory cell infiltration (mainly mononuclear cells) after 24 h and 48 h of exposure. Ear swelling was also noticed when treated with 73 ␮g CYN ml−1 (Stewart et al., 2006a). It cannot be ruled out that similar effects can occur in human

Fetal toxicity of CYN has been shown by Rogers et al. (2007) on CD-1 (Swiss-Webster) mice treated with 50 ␮g kg−1 of CYN (intraperitoneal injection) on gestasional days 8–12. Significant number of fetal deaths or earlier birth, lower number of survived pups and reduced postnatal growth of male pups was noted in treated litter. Blood in the gastrointestinal tract and hematomas in the tips of the tails occurred within survived pups. No significant differences with control in pup weight was observed. Reproductive toxicity has also been shown on human granulose cells obtained from the ovaries of women undergoing in vitro fertilization. Observed effects included inhibition of basal progesterone, main female hormone supporting gestation, occurring after 24 h exposition to 0.0625 ␮g CYN ml−1 . hCG-stimulated progesterone production decreased after 6 h exposure to 1 ␮g CYN mL−1 (Young et al.,

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2008). However, similar studies in granulose cells obtained from women with no known infertility revealed that none of used CYN concentrations (0.04–1.25 ␮g ml−1) affected basal and hCG-stimulated progesterone and estrogen production after 24 h of exposure (Young et al., 2012). More studies are necessary to investigate the effects of CYN on pregnancy.

5.

Conclusion

The expansion of CYN synthesizable cyanobacteria species is observed in Europe. Although C. raciborskii has been detected for the first time in Europe in 1938, it has been reported to spread across the continent in recent two decades. Although, some studies indicate that C. raciborskii strains identified for example in Poland does not produce this toxin, numerous other species are able to synthesize it. CYN has been detected in Europe since 2000 onwards. Impact of this toxin on human health is still a subject of investigation. Although studies that involved human cell lines were conducted and revealed a variety of CYN toxic properties, there are areas of study that are poorly investigated. So far, identified effects of CYN includes hepatotoxicity, genotoxicity, dermatotoxicity and fetal toxicity. Wide spectrum of cytotoxicity was also reported in many studies and CYN effect on immune response cannot be ruled out. Climate change and anthropogenic water eutrophication progress can have a significant role in expansion of toxic cyanobacteria. New locations of toxic species as well as CYNproducing strains may occur and raise human health concern. Therefore, water resources used recreationally and as a source of drinking water and livestock should be regularly monitored. Medical community should be aware of potential water-linked symptoms caused by exposition to cyanotoxins and report them to the designated sanitary and epidemiological reporting station.

Conflict of interest statement None.

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