In vitro investigations on uptake and

Transcription

1 In vitro investigations on uptake and toxicity of cyanobacterial toxins Dissertation Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften an der Universität Konstanz Fachbereich Biologie vorgelegt von Andreas Fischer Tag der mündlichen Prüfung: Referent: Prof. Dr. Daniel Dietrich Referent: Prof. Dr. Karl-Otto Rothhaupt Referent: Prof. Dr. Christof Hauck

2 2

3 Publications & Presentations Peer reviewed articles Fischer, A., Hoeger, S. J., Feurstein, D., Stemmer, K., Knobeloch, D., Nussler, A., and Dietrich, D. (2010). The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells. Toxicol Appl Pharmacol, in press. Fischer, A. and Dietrich, D. (in preparation). Inhibitory capacity of Adda on protein phosphatase 1 and 2A. Fischer, A., Feurstein, D., Knobeloch, D., Nussler, A., and Dietrich, D. (in preparation). In vitro toxicity of cylindrospermopsin on primary human hepatocytes and OATP-expressing HEK293 cells. Fischer, A., Hoeger, S. J., Fastner, J., Robertson, A., and Dietrich, D. (in preparation). Detection of microcystins and β-n-methylamino-l-alanine in bluegreen algae supplements. Feurstein, D., Holst, K., Fischer, A., and Dietrich, D. R. (2009). Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells. Toxicol Appl Pharmacol 234, Book section / Conference proceedings Dietrich, D. R., Fischer, A., Michel, C., and Hoeger, S. J. (2008). Toxin mixture in cyanobacterial blooms - a critical comparison of reality with current procedures employed in human health risk assessment. In Proceedings of the Interagency, International Symposium on Cyanobacterial Harmful Algal Blooms. (K. H. Hudnell, Ed.), pp Advances in Experimental Medicine & Biology. Abstracts & Poster presentations Fischer, A., Hoeger, S. J., Feurstein, D. J., Ernst, B., and Dietrich, D. R. (2007). Importance of organic anion transporting polypeptides (OATPs) for the toxicity of single microcystin congeners in vitro. 7th International Conference on Toxic Cyanobacteria, Rio das Pedras, Rio de Janeiro State, Brazil. Feurstein, D., Fischer, A., and Dietrich, D. (2007). In vitro toxicity of microcystins in primary murine whole brain and neuronal cultures. 7th International Conference on Toxic Cyanobacteria, Rio das Pedras, Rio de Janeiro State, Brazil. Feurstein, D., Fischer, A., and Dietrich, D. (2008). Microcystin congener-specific in vitro neurotoxicity. Toxicology Letters 180,

4 Contents 1 General Introduction Cyanobacteria Systematics Ecology, Morphology and Physiology Toxic Cyanobacterial Blooms and Monitoring Cyanobacterial Toxins Oligopeptides Microcystins Nodularins Alkaloids Cylindrospermopsins Saxitoxins Anatoxins Other Cyanobacterial Toxins β-n-methylamino-l-alanine Lipopolysaccharides Objectives Inhibitory capacity of Adda on protein phosphatase 1 and 2A Abstract Introduction Material & Methods Results Discussion The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells Abstract Introduction Material & Methods Results Discussion Supplemental information

5 5 In vitro toxicity of cylindrospermopsin in primary human hepatocytes and OATPexpressing HEK293 cells Abstract Introduction Material & Methods Results Discussion Detection of microcystins and β-n-methylamino-l-alanine in blue-green algae supplements Abstract Introduction Material & Methods Results Discussion General Discussion Summary Zusammenfassung Abbreviations References Apendix

6 Chapter I General Introduction 1 General Introduction 1.1 Cyanobacteria Systematics The phylum Cyanobacteria belongs to the superkingdom Eubacteria and is systematically classified into the non-filamentous orders Chroococcales and Pleurocapsales and the filamentous orders Oscillatoriales, Nostocales and Stigonematales (Castenholz and Waterbury, 1989; van den Hoek et al., 1993). A newer classification according to the National Center for Biotechnology Information (NCBI, Taxonomy Browser: Cyanobacteria, retrieved on March 17 th, 2009) includes two additional orders: Gloeobacterales and Prochlorales Ecology, Morphology and Physiology The gram-negative cynaobacteria constitute the most diverse and widespread of the phototrophic prokaryotes (Skulberg et al., 1993; Codd, 1995). They represent a considerable proportion of the marine phytoplankton and play a crucial role in photosynthetic primary production and nitrogen fixation (van den Hoek et al., 1993; Paerl, 2000). Cyanobacteria occur worldwide in nearly any given habitat. They can be found in wetlands and arid deserts, in hot springs and glaciers, even in arctic ponds and ice. However, the majority inhabits salt-, brakish- and in particular freshwater. In addition, they often represent the pioneer organisms that colonize bare areas of rock and soil (van den Hoek et al., 1993; Mur et al., 1999; Hitzfeld et al., 2000; Oliver and Ganf, 2000; Oren, 2000; Pentecost and Whitton, 2000; Vincent, 2000; Ward and Castenholz, 2000; Wynn-Williams, 2000). Their basic algal-like morphology is as diverse as their habitats: It comprises unicellular, pseudoparenchymatic, colony forming and filamentous forms with branched and unbranched trichomes (Skulberg et al., 1993; Mur et al., 1999; Whitton and Potts, 2000; Graham et al., 2009). 6

7 Chapter I General Introduction Cyanobacteria are considered not only one of the oldest life forms on earth, as fossils were dated back to 3.3 to 3.5 billion years ago (Schopf and Packer, 1987), but also the very first oxygenic photosynthesizers. This ability led to the formation of an oxygenic atmosphere, hence, paving the way for obligat aerobic prokaryotes and especially for eukaryotes (van den Hoek et al., 1993; Graham et al., 2009). Unlike eukaryotic plants, cyanobacterial thylakoids are freely located in the cytoplasm arranged concentrically and equidistantly near the cell periphery and are typically not stacked (van den Hoek et al., 1993; Mur et al., 1999; Graham et al., 2009). The accessory pigments are imbedded in phycobilisomes on the surface of the thylakoids (van den Hoek et al., 1993; Mur et al., 1999; Graham et al., 2009). In addition to chlorophyll a and carotenoids they take advantage from the use of further accessory pigments, especially phycoerythrin, phycocyanin and allophycocyanin to perform oxygenic photosynthesis. These phycobiliproteins absorp light in the range of 400 and 700 nm, thus, include the green light ( nm) that is inaccessable for green algae. Hence, they are able to live in the shadow of other phytoplankton and/or under limited light conditions (van den Hoek et al., 1993; Mur et al., 1999). Moreover, cyanobacteria are capable of altering the constituency of the phycobiliproteins and the size of the light harvesting antennae in dependence to light quality and other environmental influences. This chromatic adaptation enables them to absorp light even more efficiently (Mur et al., 1999; Oliver and Ganf, 2000; Graham et al., 2009). In order to avoid light inhibition or competitive nutrient limitation many cyanobacterial species are capable of regulating buoyancy by generation and degeneration of gas vesicles, cytoplasmatic cylindrical inclusions whose protein walls are only permeable to gases, or by accumulation of assimilation products that cause an increase in density and / or collapse of those gas vesicles as a result of invreased turgor pressure (van den Hoek et al., 1993; Oliver and Ganf, 2000; Graham et al., 2009). In stratified, non-circulating water bodies buoyancy regulation allows planktonic species to vertically adjust their position and move within the water column. This in turn enables them to exploit less competitive and nutrient rich niches like the metalimnion, where nutrients often accumulate due to the sudden change in density (Mur et al., 1999; Oliver and Ganf). A 7

8 Chapter I General Introduction further prerequisite for the formation of a metalimnetic population is a euphotic metalimnion (Lampert and Sommer, 1999; Oliver and Ganf, 2000). Moreover, these nutrients, as well as metabolites may be very effectively stored as e.g. cyanophycean starch, lipid globules, cyanophycin granules, polyphosphate bodies, carboxysomes, etc., enabling cyanobacteria to outlast temporary nutritional poverty (van den Hoek et al., 1993; Oliver and Ganf, 2000; Graham et al., 2009). Under adverse conditions, e.g. at the end of the growing season, vegetative cells of filamentous cyanobacteria with the exception of species in the order Oscillatoriales may be differentiated into akinetes, thickwalled resting cells containing storage granules. These cells are able to outlast years of dormancy (van den Hoek et al., 1993; Mur et al., 1999; Graham et al., 2009). In fact, Aphanizomenon flos-aquae and species of the genus Anabaena may stay viable in anoxic sediments for up to 18 and 64 years, respectively (van den Hoek et al., 1993). Most akinete forming species are also capable of producing heterocysts, cells specialized in nitrogen fixation that posess likewise thick walls and a hyaline protoplast. Heterocysts completely lack the oxygen-generating photosystem II and deplete diffusing oxygen by enhanced respiration in order to protect the contained nitrogenase, the highly oxygen-susceptible enzyme system that reduces atmospheric dinitrogen to ammonium ion (van den Hoek et al., 1993; Mur et al., 1999; Oliver and Ganf, 2000; Graham et al., 2009) Toxic Cyanobacterial Blooms and Monitoring As a result of their high diversity, adaptability and specialization cyanobacteria may gain competitive advantage over other photoautotrophic organisms. Thus, under favored conditions cyanobacteria often become the dominant phytoplankton of surface waters and form blooms, mass developments of one or several cyanobacterial species capable of buoyancy regulation. Blooms occur especially in eutrophic waterbodies and are an increasingly observed phenomenon due to anthropogenic nutrient input like sewage water, phosphate containing detergents and fertilizers (Bartram et al., 1999; Mur et al., 1999; Oliver and Ganf, 2000). However, mass developments may also occur in mesoand oligotrophic waterbodies: e.g. Planktothrix rubescens forms metalimnetic blooms in moderately nutrient-rich lakes (Lampert and Sommer, 1999; Mur et 8

9 Chapter I General Introduction al., 1999; Chorus, 2001). In addition, although poorly understood climatic changes, especially global warming, are considered to favour occurence, persistence and distribution of cyanobacterial blooms (Paul, 2008). Besides aesthetic nuisance caused by surface scum, discolouration of the water, bad taste and odour blooms may pose a serious threat for human and animal health as they may contain highly potent toxins (Bartram et al., 1999; Kuiper-Goodman et al., 1999). Amongst a total of over 150 cyanobacterial genera including approximately 2,000 species about 40 toxigenic species are known (Skulberg et al., 1993). Although this represents a rather small proportion it was estimated that approximately 25-75% of bloom isolates are capable of producing toxins (Lawton and Codd, 1991; Chorus, 2001). This demonstrates the widespread distribution of toxigenic species and hence the importance to monitor surface waters where cyanobacterial blooms regularly occur, especially those used as a drinking water source or for recreational purposes. However, monitoring is hampered by the highly variable nature of blooms: A bloom may be dominated by a single species or be contemporaneously composed of several species of which some or the majority may be non-toxic. Even within a certain species there may be a variety of toxic and non-toxic strains at the same time (Vezie et al., 1998; Sivonen and Jones, 1999; Pereira et al.; Moreno et al., 2004; Molica et al., 2005). Thus, there is a high risk to overlook toxigenic species, especially when monitoring is based on species identification only. Naturally phytoplankton communities are never static in their composition. Instead they are usually characterized by a seasonal succession, i.e. the species composition is changing during the course of seasons (Lampert and Sommer, 1999). This applies for cyanobacteria as well: in a single waterbody blooms of different cyanobacterial species have been reported to occur. Usually those blooms are temporally seperated or in succession, but have occasionally been found to be overlapping, too (Carmichael et al., 2000; Pereira et al., 2000; Hoeger et al., 2004). Accordingly, seasonal changes in toxin production occur as well. However, size and density of a bloom do not necessarily correlate with the amount of toxin produced, thus, are not a reliable indicator for the presence 9

10 Chapter I General Introduction of toxins (Watanabe et al., 1992; Vezie et al., 1998; Sivonen and Jones, 1999; Hoeger et al., 2004; Dietrich et al., 2008). Besides seasonal changes in the abundance of cyanobacterial populations and toxins their spacial distribution may vary as it succumbs different influences like surface and underwater topography, stratification, currents and wind (Watanabe et al., 1992; Lampert and Sommer, 1999; Oliver and Ganf, 2000; Falconer, 2005a). Consequently, both temporal and spacial distribution of cyanobacterial populations and hence the toxins potentially produced have to be taken into account for the determination of monitoring sites and frequency. 1.2 Cyanobacterial Toxins Cyanobacteria produce a multitude of secondary metabolites, whose physiological functions and ecological regulations remained predominantly unknown to date. However, some of them revealed to be toxic towards aquatic and terrestrial organisms, especially mammals (Sivonen and Jones, 1999; Dow and Swoboda, 2000; Kaebernick and Neilan, 2001; Welker and von Dohren, 2006). Several different toxins and toxin congeners may be simultaneously produced by a single species or strain (Harada et al., 1991a; Sivonen et al., 1992; Park et al., 1993; Sivonen and Jones, 1999; Harada et al., 2001; Welker and von Dohren, 2006). In addition, cyanotoxin production appears to be variable and facultative, i.e. its pattern may alter qualitatively and quantitatively in a certain strain or species in response to several environmental and physiological factors (Sivonen et al., 1995; Rapala et al., 1997; Sivonen and Jones, 1999; Kaebernick and Neilan, 2001). Most cyanotoxins are intracellular toxins and only marginally excreted. Their predominant release in the environment naturally occurs during cell scenescence, death and lysis. Thus, as long as toxigenic blooms are healthy, extracellular toxin concentrations remain low until its decay or artificial lysis after application of algicides (e.g. copper sulphate) (Jones and Orr, 1994; Kuiper- Goodman et al., 1999; Sivonen and Jones, 1999). 10

11 Chapter I General Introduction Cyanotoxins are usually classified according to their chemical structure, toxicity or organ specifity. Since the latter two often appear to have quite some diversity depending on the respective toxin and the route of administration the following classification of the most important cyanotoxins uses a structural approach, whereas the main focus lies on those that were investigated in this study Oligopeptides Oligopeptides represent the major part of cyanobacterial secondary metabolites (Welker and von Dohren, 2006). In cyanobacterial blooms of fresh and brackish waters the cyclic microcystins and nodularins are globally the most frequently occuring cyanotoxins of this class and turned out to be the most toxic at the same time (Sivonen and Jones, 1999; Spoof, 2005). Further oligopeptides frequently produced by bloom-forming cyanobacteria include cyanopeptolins, anabaenopeptins, microviridins, microginins and aeroginosins. However, these inhibitors of serine proteases, serine/threoninespecific protein phosphatases and other enzymes revealed to be far less toxic (Kaya et al., 1996; Namikoshi and Rinehart, 1996; Sano et al., 2001; Hastie et al., 2005; Ersmark et al., 2008; Sedmak et al., 2008) Microcystins Microcystins (MCs) are cyclic heptapeptides that have first been isolated from their eponymous producer Microcystis aeruginosa (Bishop et al., 1959; Konst et al., 1965; Carmichael et al., 1988a). Further species of the genus Microcystis, as well as Anabaena, Planktothrix, Oscillatoria, Nostoc, Anabaenopsis, Radiocystis, Arthrospira and Hapalosiphon have been reported to produce MCs (Sivonen and Jones, 1999; Spoof, 2005). Structure The general structure of MCs (Fig. 1.1) is cyclo(-d-ala 1 -L-X 2 -D-erythro-βmethylAsp 3 -L-Z 4 -Adda 5 -D-Glu 6 -N-methyldehydro-Ala 7 ) in which Adda stands for the unique D-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid and X and Z for variable L-amino acid residues (Botes et al., 1984; Botes et al., 1985; Rinehart et al., 1988; Rinehart et al., 1994). Substitutions in 11

12 Chapter I General Introduction those two positions constitute the main structural variations and are therefore used for the nomenclature of MCs (e.g. MCLR is a microcystin congener with L- leucine and L-arginine in position 2 and 7, respectively) (Carmichael et al., 1988a). However, further variations (e.g. demethylation of D-erythro-βmethylaspartic acid (D-MeAsp) and N-methyldehydroalanine (Mdha)) may occur in any of the seven amino acids leading to more than 80 structural analogues with molecular weights ranging from 900 to 1100 Da (Sivonen and Jones, 1999; Spoof, 2005; Zurawell et al., 2005; Humpage, 2008). Fig. 1.1: General structure of microcystins. Synthesis MCs are synthesized non-ribosomally by peptide synthetases and polyketide synthases that are combined in a multi-enzyme complex. A single gene cluster with ten open reading frames encodes for modules (mcya - mcyj) which compose the enzymes for MC synthesis. Nine modules have a synthesizing function, whereas one (McyH), a transmembrane protein belonging to the ATPbinding cassette transporter family, is putatively responsible for toxin transport and/or localization (Moore et al., 1991; Rinehart et al., 1994; Tillett et al., 2000; Falconer, 2005a; Welker and von Dohren, 2006). However, McyH seemed to be additionally involved in the microcystin biosynthesis pathway, since its deletion led to a complete halt in MC production (Pearson et al., 2004). Moreover, a multiplex polymerase chain reaction has been developed that can be used to identify contamination with microcystin producing cyanobacteria in 12

13 Chapter I General Introduction cyanobacterial dietary supplements and possibly other food products by amplification of genes like mcya of the microcystin synthetase gene cluster (Saker et al., 2005; Saker et al., 2007). In general, several MC congeners are produced at the same time (Harada et al., 1991b; Sivonen et al., 1992; Luukkainen et al., 1993; Namikoshi et al., 1995; Edwards et al., 1996; Lawton et al., 1999; Spoof, 2005; Welker and von Dohren, 2006; Pegram et al., 2008). Degradation The cyclic structure of MCs appears to be extremely stable and insusceptible towards temperature, ph, chemical hydrolysis and oxidation, especially under natural environmental conditions (Harada et al., 1996a; Harada and Tsuji, 1998). Hence, MCs may persist and remain toxic in waterbodies for weeks until slow photochemical degradation which is significantly accelerated in the presence of pigments (Jones et al., 1994; Tsuji et al., 1995; Lahti et al., 1997; Sivonen and Jones, 1999). Besides, certain heterotrophic aquatic bacteria of different genera (e.g. Sphingomonas, Pseudomonas and Paucibacter) are capable of decomposing MCs after an initial lag phase of several days. Depending on different environmental factors and MC concentrations complete or major degradation has been shown to occur within 2-10 days (Rapala et al., 1994; Bourne et al., 1996; Takenaka and Watanabe, 1997; Park et al., 2001; Christoffersen et al., 2002; Ishii et al., 2004). Toxicity and Molecular Mode of Action The majority of cyanobacterial poisonings of animals and humans are attributed to MCs. In mammals they predominantly affect the liver and hence are generally referred to as hepatotoxins (Carmichael, 1997; Kuiper-Goodman et al., 1999; Codd et al., 2005). MC toxicity predominantly relies on the very potent inhibition (at nanomolar concentrations) of serine/threonine-specific protein phosphatases (PPs) PP1 and PP2A, as well as PP3 to PP6, whereas inhibition of PP2B, PP2C and PP7 revealed to be ineffective (MacKintosh, 1993; Honkanen et al., 1994; Runnegar et al., 1995a; Toivola et al., 1997; Hastie et al., 2005). The causality between phosphatase inhibition and in vivo toxicity was demonstrated in mice (i.p.) by concomitant measurement of liver PP1 and PP2A activity and examination of 13

14 Chapter I General Introduction the clinical symptoms. Phosphatase inhibition was dose-dependent and proportional to the severity of the liver demage (Runnegar et al., 1993). The interaction between MCs and phosphatases comprises a two-step mechanism in which the first step already mediates the inhibition: An initial noncovalent, hence, reversible binding is formed within minutes by the alignment of the Adda side chain into a hydrophobic groove adjacent to the phosphatase s catalytic site and the formation of a hydrogen bond between the carboxyl group of D-glutamic acid and the binuclear metal ion catalytic centre of the phosphatase (Goldberg et al., 1995; Craig et al., 1996). An additional ionic interaction occurs between the carboxyl group of D-erythro-β-methylaspartic acid and arginine 96 and tyrosine 134 of the phosphatase s catalytic subunit (PPc) (Bagu et al., 1997; Maynes et al., 2006). In the second step that lasts several hours the methyl group of N-methyldehydroalanine is linked covalently to cysteine of the catalytic subunit of the phosphatase (cysteine 273 of PP1c and cysteine 266 of PP2Ac), which renders the binding irreversible, however, does not increase the inhibitory activity (MacKintosh et al., 1995; Runnegar et al., 1995a; Craig et al., 1996; Bagu et al., 1997; Maynes et al., 2006). A few MC congeners, as well as the closely related nodularins (see and Fig. 1.2), in which N-methyldehydroalanine is substituted by N- methyldehydrobutyrine are unable to form this covalent linkage to the phosphatases (Bagu et al., 1997; Hastie et al., 2005). This does also apply for dihydromicrocystins, whose double bond of N-methyldehydroalanine is reduced (MacKintosh et al., 1995; Craig et al., 1996). However, these modifications do not (Sano et al., 2004) or only moderately (5- to 50-fold) decrease the inhibitory potential (MacKintosh et al., 1995; Hoeger et al., 2007) and hence toxicity as shown in mice (Rinehart et al., 1994; Sivonen and Jones, 1999). On the contrary, the Adda-glutamate moiety was found to be crucial for the inhibitory capacity: MCLR and MCRR inhibited PP2A 100-times stronger than their geometrical isomers, [6(Z)-Adda 5 ]MCLR and [6(Z)-Adda 5 ]MCRR (Nishiwaki- Matsushima et al., 1991). Indeed, any structural modifications of either Adda (e.g. isomerization of its diene from 6(E) to 6(Z)) or D-glutamate (e.g. acetylation or esterification) have been reported to dramatically decrease or abolish the toxicity of MCs in mice (Harada et al., 1990a; Harada et al., 1990b; Nishiwaki-Matsushima et al., 1991; Namikoshi et al., 1992; Stotts et al., 1993; 14

15 Chapter I General Introduction Rinehart et al., 1994; Harada, 1996b). On the other hand, isolated Adda neither elicited inhibitory action on PP1 even at 10 µm (see also chapter III) nor toxicity at concentrations up to 10 mg/kg body weight (mouse, i.p.), while MCLR caused typical concentration-response effects with an IC 50 (concentration that inhibits 50% of the enzyme s activity) of 2 nm demonstrating the relevance of the remaining structural units for the biological activity (Harada et al., 2004). In general, since the Adda-glutamate moiety is very conserved variation in toxicity is low amongst most MC congeners with LD 50 values (dose of toxin that kills 50% of the exposed animals) ranging from µg/kg body weight (mouse, i.p.) (Rinehart et al., 1994; Sivonen and Jones, 1999). An exception is MCRR whose LD 50 (600 µg/kg body weight (mouse, i.p.)) appeared to be approximately one order of magnitude higher than MCLR (Krishnamurthy et al., 1986; Watanabe et al., 1988). Surprisingly, the inhibitory activity of both congeners on PP2A revealed to be in the same range: the IC 50 of MCRR and MCLR were 3.4 nm and 1.6 nm, respectively (Yoshizawa et al., 1990; Fujiki et al., 1996). Consequences of Phosphatase Inhibition PPs catalyze the dephosphorylation of intracellular phosphoproteins, thus represent the antagonists of protein kinases. Their interplay allows for concerted regulation of enzymes and other proteins which in turn regulate or control a vast variety of cellular functions and processes. PPs of type 1 and type 2 occur in all eukaryotes where they are responsible for the dephosphorylation of serine and threonine residues, thus a plethora of target proteins. E.g. PP1 and PP2A play pivotal roles in the regulation of cell growth and division, metabolism (e.g. glycogen metabolism), muscle contraction, intracellular transport, gene expression and protein synthesis (Cohen and Cohen, 1989a; Cohen, 1989b; Bollen and Stalmans, 1992; MacKintosh, 1993; Mumby and Walter, 1993; Cohen, 2002). Consequently, the inhibition of these PPs by MCs, which is also referred to as an activation of the corresponding protein kinases, results in the perturbance and disregulation of the listed cellular functions. In general, the eqilibrium between dephosphorylation and phosphorylation displaces, leading to an overall increase in phosphorylated cytosolic and cytoskeletal phosphoproteins as observed in isolated rat hepatocytes (Yoshizawa et al., 1990; Eriksson et al., 15

16 Chapter I General Introduction 1990a; Falconer and Yeung, 1992a). This MC-induced hyperphosphorylation was observed in all cytoskeletal components, i.e. microfilaments, microtubules and intermediate filaments (especially keratin 8 and 18) and resulted in their rapid reorganization and loss of cell integrity (Ohta et al., 1992; Wickstrom et al., 1995; Toivola et al., 1997; Toivola et al., 1998). Batista et al. (Batista et al., 2003) made similar observations with primary human hepatocytes, whose actin mesh collapsed into the centre of the cell following treatment with MCLR. MC-induced Apoptosis Depending on dose and time, either necrosis (at high concentrations) or apoptosis (at lower concentrations) has been observed subsequent to the disruption of the cytoskeleton (Hooser et al., 1991; McDermott et al., 1998; Hooser, 2000; Batista et al., 2003). However, the molecular mechanisms of MC-induced apoptosis are not entirely elucidated, although PP inhibition appeared to be crucial in triggering or executing programmed cell death (Fladmark et al., 2002). An additional role has been attributed to MC-induced generation of reactive oxygen species (ROS) causing mitochondrial permeability transition, a critical event in the progression of apoptotic cell death (Ding et al., 2000; Ding and Nam Ong, 2003; Gehringer, 2004a; Weng et al., 2007). Mikahailov et al. (Mikhailov et al., 2003) identified the ATP-synthase beta subunit as a further yet less important molecular target of MCs. They hypothesized that the adduct formation with MCs at high concentrations might play a mechanistic role in MC-induced apoptotic signalling by causing mitochondrial damage, i.e. loss of mitochondrial membrane potential and perturbance of mitochondrial functions. Acute and Subacute Effects The effects and symptoms of intoxications with MCs are as manifold as the consequences of PP inhibition may suggest. However, their severity depends on many factors like dose and duration of the exposure, as well as the route of intoxication and may vary among different species, gender and age (Dietrich and Hoeger, 2005; Fournie and Hilborn, 2008). Acute exposure to high doses of MCs causes sinusoid disruption, hepatocyte deformation and necrosis followed by rapid death (1-3 hours in mice) from liver haemorrhage or from liver failure (Falconer et al., 1981; Runnegar and 16

17 Chapter I General Introduction Falconer, 1982; Runnegar et al., 1986; Theiss et al., 1988; Hooser et al., 1989; Beasley et al., 2000). Hepatic and endothelial lesions are thereby accompanied by an increase in liver weight and size, as well as serum liver enzyme levels. Further pathological and ultrastructural features diagnosed in the liver are centrilobular hepatic necrosis, cessation of bile flow, loss of microvilli, bleb formation and induction of apoptosis in hepatocytes (Runnegar et al., 1995b; Wickstrom et al., 1996; Ito et al., 1997; Yoshida et al., 1997). Other organs affected, albeit less severely, include stomach, intestine, kidneys and lungs (Runnegar et al., 1986; Hooser et al., 1989; Falconer et al., 1992b; Falconer and Humpage, 1996; Ito et al., 1997). Furthermore, oral MC toxicity has also been shown to depend on the nutritional state of the exposed animals: fed rats were 1.7-fold less susceptible than fasted rats (25-h i.p. LD 50 of 72 µg/kg bw) (Miura et al., 1991). The authors suggested the higher susceptibility to either stem from the additional depletion of the already exhausted glycogen stores in fasted rats by activation of phosphorylase a as a result of MC mediated PP inhibition impairing the animal s energy reserves or the decreased respiratory capacity in fasted rats leading to a more advanced mitochondrial damage. A multitude of symptoms have been documented from acute human intoxications: In 1996, in a haemodialysis unit in Caruaru, Brazil, water contaminated with MC (and possibly cylindrospermopsin) was used for dialysis. 116 out of 131 patients developed symptoms of acute neuro- and hepatotoxicity including visual disturbances, vertigo, headaches, nausea and vomiting, muscle weakness and myalgia, painful huge hepatomegaly, liver plate disruption, liver cell deformity, necrosis and apoptosis, as well as death from liver failure. Biochemical investigations showed elevated liver enzyme activities, severe hypertriglyceridaemia and hyperbilirubinaemia. 52 patients succumbed to the so-called Caruaru syndrome (Jochimsen et al., 1998; Pouria et al., 1998; Kuiper-Goodman et al., 1999; Carmichael et al., 2001; Azevedo et al., 2002). In 1988, an incident of acute oral intoxication via contaminated drinking water led to a severe gastro-enteritis epidemic (about 2,000 cases) in the area of the Itaparica Dam, Bahia, Brazil which resulted in 88 deaths of predominantly children under the age of 5. The symptoms most commonly observed included 17

18 Chapter I General Introduction diarrhoea, colic-like abdominal pain, vomiting and fever. The newly flooded dam accomodated an immense cyanobacterial bloom of the genera Anabaena and Microcystis and the cases of gastro-enteritis were restricted to areas in which the dam served as a drinking water source (Teixera et al., 1993). Subacute oral intoxications with MCs at lower concentrations are characterized by diarrhoea, vomiting, weakness, pallor and elevated levels of hepatic enzymes in plasma which indicate toxic liver injury (Falconer et al., 1983; Bell and Codd, 1994). Chronic Effects and Tumour Promotion Chronic exposure to low doses of MCs has been shown to promote tumours in humans and animals. Falconer et al. (Falconer et al., 1988) examined the effects of exposure of mice to a toxic extract of Microcystis aeruginosa via drinking water over a period of 1 year. At high concentrations 4 out of 71 mice developed tumours, in contrast to only 2 out of 223 mice at lower concentrations. An epidemiological study on the incidence of primary liver cancer (PLC) in China, which is one of the highest worldwide with 24 mortalities per 100,000 population, revealed strongest correlations with hepatitis B incidence, followed by aflatoxins in the diet and MC contaminated drinking water from ponds and ditches. All three factors are considered to act together in promoting PLC (Yu, 1989; Yu, 1995; Falconer et al., 1999; Kuiper-Goodman et al., 1999). Since phosphatases may act as tumour suppressors, tumour promotion is possibly a result of phosphatase inhibition leading to MAPK signaling which in turn stimulates proliferation and inhibits apoptosis (Toivola and Eriksson, 1999; Gehringer, 2004a). Indeed, several tumour-promoting toxins like okadaic acid, calyculins, tautomycin, as well as nodularins and MCs are known to act via inhibition of PP1 and PP2A (MacKintosh, 1993). Evidence for the tumourpromoting and -initiating activity of MCs have been provided by several in vitro and in vivo studies. Suppression of apoptosis and stimulation of cytokinesis has been reported at lower MC concentrations (pm range) in polyploid hepatocytes in vitro (Humpage and Falconer, 1999). In a two-stage carcinogenesis study MCLR dose-dependently increased the occurence of positive foci of the placental form of glutathione S-transferase in rat liver initiated with diethylnitrosamine (Nishiwaki-Matsushima et al., 1992). Without initiator 18

19 Chapter I General Introduction neoplastic nodules formed in mice liver after repeated (100 times) i.p. injections of a sublethal dose (20 µg/kg bw) of MCLR. However, neither nodule formation nor liver damage was observed when MCLR (80 µg/kg bw) was orally administered (Ito et al., 1997). The genotoxic potential of MCs has been furthermore assessed in several genotoxicity assays. In human HepG2 cells MCLR dose- and time-dependently induced DNA strand breaks (Zegura et al., 2003; Zegura et al., 2004), whereby this effect could be reduced by different ROS scavengers. The authors therefore concluded that MCLR causes DNA damage by inducing the formation of ROS. Carcinogenic effects were also supported by Sano et al. (Sano et al., 2004): They reported the development of spontaneous liver tumour in 15 out of 22 mice i.p. injected with MCLR (12.5 or 25 µg/kg bw) once a week for 14 months. Tumour incidences thereby correlated with the generation of 8- hydroxydeoxyguanosine, a biomarker for oxidative stress, in the liver of the mice. In addition, in the in vitro cytokinesis-block micronucleus (CBMN) assay, a test that detects both chromosome loss and chromosome breakage, MCLR failed to induce significant alterations of DNA in contrast to nodularin and okadaic acid (Fessard et al., 2004). Thus, MCs appear not to be directly genotoxic, but indirectly by generating ROS at moderate to high concentrations. Moreover, for liver tissue damage a no observed adverse effect level (NOAEL; the highest concentration that fails to elicit signs of adverse effects) of 40 µg MCLR/kg bw per day was estimated from a subchronic study in which mice were orally gavaged with pure MCLR over a period of 13 weeks (Fawell et al., 1999). This NOAEL was employed with additional uncertainty factors (10 a total of 1,000) to derive a provisional tolerable daily intake (TDI) of 0.04 µg MCLR/kg bw (Falconer et al., 1999; Kuiper-Goodman et al., 1999; Dietrich and Hoeger, 2005), which has been used as a basis for risk assessments and calculations of guideline values including drinking water (1 µg/l; WHO, 1998) and cyanobacterial dietary supplements (1 µg/g dw; Gilroy et al., 2000). Extract Toxicity and Synergistic effects Surprisingly, the toxicity of cyanobacterial extracts often exceeds the toxic potential that would have been expected from the contained amount of toxins. This phenomenon probably relies on unknown or unnoticed active compounds 19

20 Chapter I General Introduction that are additionally contained in the extracts or on toxins acting synergistically as recently discussed (Dietrich et al., 2008; Pegram et al., 2008). Indeed, Fitzgeorge et al. (Fitzgeorge et al., 1994) determined an intranasal LD 50 of 2000 µg/kg bw for anatoxin-a (see ) in mice that was lowered to 500 µg/kg bw when a sublethal dose of 31.3 µg MCLR/kg bw (NOAEL for liver weight increase) was administered 30 minutes prior to anatoxin-a. By contrast, this synergism failed to recur by oral application of the toxins which was suggested to be due to the different route of administration (Rogers et al., 2005). Routes of Intoxication Intoxications with cyanobacterial toxins may occur via different routes of exposure as previously described (Falconer et al., 1999; Dietrich and Hoeger, 2005; Dietrich et al., 2008). Such scenarios include: exposure via contaminated drinking water exposure via contaminated food as a result of bioaccumulation in the food chain, irrigation with contaminated water or toxic blooms in rice fields exposure from recreational use of water exposure from contaminated cyanobacterial dietary supplements exposure via renal dialysis. The case studies on human poisonings described above demonstrate both the high symptomatic diversity of MC intoxications and their dependency on the respective route of intoxication that was elucidated in various animal studies as well: In mice the i.p. LD 50 of MCLR or Microcystis extracts appeared to be approximately a factor lower than the oral LD 50 (Falconer, 1991; Kotak et al., 1993; Yoshida et al., 1997; Fawell et al., 1999). In contrast, other routes of exposure of mice to MCLR corresponded well to the lethal dose by i.p. application with LD 50 s between 50 and 100 µg/kg bw for intratracheal (Ito et al., 2001), 43 µg/kg bw for intranasal and 67 µg/kg bw for i.v. application (Creasia, 1990). Differences in the LD 50 s and the symptoms elicited reflect varying bioavailability from the respective route of administration as a result of the chemical and biochemical characteristics of MCs and their toxicokinetics, i.e. transport and distribution in the exposed organism as specified in the following. 20

21 Chapter I General Introduction Organotropism, Uptake, Distribution and Excretion Cellular trafficking of MCs requires active transport, since they are rather hydrophilic molecules which precludes passive diffusion through cell membranes. The selective uptake into hepatocytes via the bile acid transport system has been demonstrated by its inhibition and by coincubation with bile salts and further substrates of this transport system, which reduced MC uptake and toxicity (Eriksson et al., 1990b; Runnegar et al., 1991; Runnegar et al., 1995c). Indeed, Fischer et al. (Fischer et al., 2005) identified members of the multispecific organic anion transporting polypeptides [human: OATPs/SLCOs; animals: Oatps/Slcos; (protein name/gene symbol) (Hagenbuch and Meier, 2004)], which are part of the bile acid transport system, as being capable of transporting [ 3 H]-dihydro-MCLR. Those members included OATP1B1, OATP1B3 and Oatp1b2 (rat), all located at the basolateral (sinusoidal) membrane of hepatocytes, as well as OATP1A2, located in liver, kidney and at the blood-brain-barrier (Hagenbuch and Meier, 2003; Hagenbuch and Meier, 2004; Bronger et al., 2005; Ho and Kim, 2005; Nies, 2007). Exchange with anions (e.g. bicarbonate) or efflux of glutathione and/or glutathione-s-conjugates is assumed to be the driving force for OATP/Oatpmediated transport as demonstrated in rat Oatp1a1 and -1a4 (Satlin et al., 1997; Li et al., 1998; Hagenbuch and Meier, 2003; Hagenbuch and Meier, 2004; Ho and Kim, 2005). Monks et al. (Monks et al., 2007) and Komatsu et al. (Komatsu et al., 2007) confirmed the uptake of non-labeled MCLR and further congeners via OATP1B1 and OATP1B3. Therefore, it has been suggested that OATP1B1, OATP1B3 and OATP1A2 are at least involved in the observed MC-mediated hepato- and neurotoxicity (Fischer et al., 2005; Dietrich et al., 2008). The organotropism was additionally corroborated by various investigations using radiolabeled MCs and different application routes as summarized by Dietrich and Hoeger (Dietrich and Hoeger, 2005). Briefly, radioactivity was predominantly detected in the liver, followed by the gastro-intestinal tract, the kidneys, the brain, the lungs and other organs. Recovery of radiolabeled MC after oral administration was thereby tremendously reduced (factor 80) in comparison to i.p. or i.v. injection indicating reduced bioavailability from oral application. Indeed, following oral ingestion it has been shown in mice that a 21

22 Chapter I General Introduction significant portion of free MC is not absorbed, but remains in the gastrointestinal tract and thus is likely to be excreted via faeces (Fujiki et al., 1996; Ito et al., 2000). Absorption was demonstrated in animal studies to mainly take place in the small intestine, especially in the ileum, but also to a small extend in the stomach (Dahlem et al., 1989; Stotts et al., 1997a; Stotts et al., 1997b; Ito et al., 2000). Subsequently, MC enters the venous blood stream, following the portal vein to the liver, where it rapidly accumulates (Stotts et al., 1997a; Stotts et al., 1997b) due to the first pass effect and selective uptake via the bile acid transport system, which was also observed after i.v. or i.p. injection (Falconer et al., 1986; Brooks and Codd, 1987; Robinson et al., 1989; Meriluoto et al., 1990; Robinson et al., 1991; Lin and Chu, 1994). Although the vast majority of MC is retained in the liver, it was also detectable in the bile (Runnegar et al., 1986; Stotts et al., 1997a; Stotts et al., 1997b), as well as in intestines and feces following i.v. and i.p. injection (Robinson et al., 1989; Robinson et al., 1991; Stotts et al., 1997a; Wang et al., 2008) providing evidence for enterohepatic circulation. Besides biliary excretion, elimination of MCs also occurs via urine, albeit to a much lesser extent (Falconer et al., 1986; Runnegar et al., 1986; Robinson et al., 1989; Robinson et al., 1991). Metabolization and Detoxification MCs are not degraded in the mammalian digestive tract due to the predominant presence of D-amino acids and the cyclic structure, which renders them resistant to enzymatic hydrolysis by eukaryotic peptidases (e.g. trypsin) (Runnegar and Falconer, 1981; Harada and Tsuji, 1998). However, strong evidence for the detoxification of MCs via the glutathione (GSH) pathway was provided by Hermansky et al. (Hermansky et al., 1991): pretreatment with GSH protected mice (no mortalities were observed) against a lethal dosage of MCLR. Indeed, Kondo et al. (Kondo et al., 1996) detected several MC metabolites among which two were identified as GSH and cysteine conjugates in mice and rats. The conjugation sites were shown to be the thiols of GSH and cysteine that bind nucleophilically to the Mdha residue of MCs, identical to the PPs (Kondo et al., 1992). 22

23 Chapter I General Introduction Under physiological conditions this reaction is catalyzed by the glutahtione S- transferase (GST) as demonstrated by GST assays (Pflugmacher et al., 1998; Takenaka, 2001) and increased GST activity in mice that corresponded with increased GST transcription following treatment with MCLR (Gehringer et al., 2004b). Further evidence for the detoxification of MCs by GSH/cysteine conjugation were given by Ito et al. (Ito et al., 2002): Both MCLR-GSH and MCLR-cysteine conjugates, administered intratracheally to mice, exhibited 12-fold reduced toxicity compared to native MCLR, although they inhibited PP1 and PP2A nearly equipotently in vitro. However, immunostaining revealed the highest signals for both conjugates in the kidneys and intestines of the mice, whereas the characteristic accumulation and damage in the liver failed to occur. The authors suggested that either the uptake of the conjugates into the liver is impeded or that they are effectively exported, e.g. by the ATP-dependent glutathione S-conjugate export (GS-X/MRP1), officially known as ABCC1 according to the HUGO Gene Nomenclature Committee (HGNC). The potential involvement of members of the ATP Binding Cassette superfamily (ABC transporters), especially multidrug resistance-associated proteins (MRPs) and multidrug resistance proteins (MDRs), in the export of MCs has recently been discussed by Dietrich et al. (Dietrich et al., 2008) Nodularins The first report on fatal animal poisonings by toxic Nodularia spumugena, the eponymous producer of nodularins (NODs), has been published by Francis in the late 19 th century (Francis, 1878). The general structure of NODs (Fig. 1.2; MW = 824 Da), cyclo(-d-erythro-βmethylasp 1 -L-Z 2 -Adda 3 -D-Glu 4-2-(methylamino)-2-dehydrobutyric acid 5 ) (Rinehart et al., 1988; Carmichael et al., 1988b; Sivonen and Jones, 1999; Spoof, 2005), obviously demonstrates their close relatedness to MCs. Besides being composed of five instead of seven amino acids, i.e. D-alanine and the adjacent variable L-amino acid are missing, NODs further differ from MCs in the substitution of Mdha by methyldehydrobutyric acid (Mdhb). The variable L- amino acid Z either represents arginine in nodularin-r (often simply referred to as nodularin (Rinehart et al., 1988)) or valine in nodularin-v (trivially named 23

24 Chapter I General Introduction motuporin) which was isolated from the marine sponge Theonella swinhoei (de Silva et al., 1992), however, is likely to originate from a cyanobacterial symbiont. Aside from these two analogues, only a few further variants have been found including two demethylated variants, [D-Asp 1 ]nodularin and [DMAdda 3 ]nodularin and the non-toxic [(6Z)-Adda 3 ]nodularin (Namikoshi et al., 1994; Rinehart et al., 1994). The relatedness of NODs and MCs reflects in nearly every aspect: NODs are also synthesized non-ribosomically by peptide synthetases and polyketide synthases. Their biosynthesis gene cluster is homologous to the mcy-cluster, however, consists of only nine open reading frames (ndaa - ndai) and lacks the two modules in the MC gene cluster that are responsible for the synthesis of D- alanine and the adjacent variable L-amino acid (Moffitt and Neilan, 2004; Rantala et al., 2004; Welker and von Dohren, 2006). Fig. 1.2: General structure of nodularins. Likewise MCs the molecular mode of action of NODs is based on the inhibition of serine/threonine-specific PPs, thus, the toxic effects elicited by both cyanopeptides are similar and comparably severe (Eriksson et al., 1988; Runnegar et al., 1988; Carmichael et al., 1988b; Yoshizawa et al., 1990; Ohta et al., 1994). However, as already mentioned (see ), NODs are unable to bind covalently to the phophatases (Craig et al., 1996; Bagu et al., 1997). Nevertheless, MCLR and NOD (both NOD-R and NOD-V) equipotently inhibit 24

25 Chapter I General Introduction PP1 and PP2A (Yoshizawa et al., 1990; Honkanen et al., 1991; de Silva et al., 1992). Furthermore, NOD revealed to be a stronger tumour promoter than MCLR as it more effectively increased the occurence of positive foci of the placental form of glutathione S-transferase in rat liver initiated with diethylnitrosamine in a twostage carcinogenesis experiment. In contrast to MCLR, NOD also induced positive foci without prior initiation which classifies it as a carcinogen (Ohta et al., 1994; Fujiki et al., 1996) Alkaloids The collective term alkaloid stands for basic, nitrogenous, heterocyclic compounds that are naturally produced in the secondary metabolism of predominantly plants (Vollhardt and Schore, 1995; Nultsch, 2001; Bruice, 2007). As broad as this definition is the structural and toxicological diversity of the cyanobacterial alkaloid toxins. They comprise dermatotoxic (e.g. lyngbyatoxin-a, aplysiatoxins), neurotoxic (e.g. saxitoxins and anatoxins) and cytotoxic (e.g. cylindrospermopsins) compounds that are regularly reponsible for severe, sometimes even fatal, human and animal poisonings (Kuiper-Goodman et al., 1999; Sivonen and Jones, 1999) Cylindrospermopsins In 1979 on Palm Island located off the coast of Queensland, Australia an algal bloom occured in Solomon Dam, the main drinking water reservoir of that island, causing discolouration and bad taste and odour of the water. In order to antagonize the bloom, it was treated with copper sulphate. Subsequently, among the local Aboriginal population 139 children and 10 adults developed severe hepatoenteritis. No cases of this illness were reported from a small group of the island s population that drew their drinking water from an alternative source. Since the identification of causative pathogenes, toxins or chemicals failed, the illness was entitled Palm Island Mystery Disease (Byth, 1980; Bourke et al., 1983). Investigations on following algal blooms in Solomon Dam identified the cyanobacterium Cylindrospermopsis raciborskii as the bloom forming species 25

26 Chapter I General Introduction and the most plausible causative of the disease. Extracts of this species elicited dose-dependent damage to the livers of mice following i.p. injection. At lower concentrations (10.5 mg/kg mouse bw) hepatocyte necrosis was mainly restricted to centrilobular regions, whereas at high concentrations (168 mg/kg mouse bw) all hepatocytes appeared to be affected. An LD 50 of 64 ± 5 mg/kg bw was determined at 24 hours after administration. Although the extracts were found to be primarily hepatotoxic, lungs, kidneys and the small intestine were also affected (Hawkins et al., 1985). From this extract Ohtani et al. (Ohtani et al., 1992) isolated and characterized an unusual alkaloid with a molecular weight of 415 Da that elicited the same symptoms: cylindrospermopsin (CYN; Fig. 1.3). This highly water-soluble zwitterion consists of a sulphated and methylated tricyclic guanidino moiety that is linked to uracil via a hydroxylated carbon (Ohtani et al., 1992; Falconer, 2005a). Currently, only two naturally occuring structural variants have been identified: 7-deoxycylindrospermopsin (Norris et al., 1999) and 7- epicylindrospermopsin (Banker et al., 2000). Fig. 1.3: Structure of cylindrospermopsin. Pure CYN appeared to be relatively stable at extreme temperatures, ph and sunlight, whereas in algal extracts it degrades rapidly when exposed to sunlight probably due to several attendant pigments (Chiswell et al., 1999). Besides C. raciborskii CYN has been further isolated from Umezakia natans (Harada et al., 1994), Aphanizomenon ovalisporum (Banker et al., 1997), Raphidiopsis curvata (Li et al., 2001), Anabaena bergii (Schembri et al., 2001), Aphanizomenon flos-aquae (Preussel et al., 2006) and Aphanizomenon gracile (Rücker et al., 2007; Wiedner et al., 2008). 26