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-Ala¹-L-X² -D-erythro-β-methylAsp³-L-Z⁴-Adda⁵-D-Glu⁶-N-methyldehydro-Ala⁷) 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

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-β-methylaspartic 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 ATP-binding 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

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

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 non-covalent, 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 1.2.1.2 and Fig. 1.2), in which 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)-Adda⁵]MCLR and [6(Z)-Adda⁵]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;

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 IC50 (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 LD50 values (dose of toxin that kills 50% of the exposed animals) ranging from 50 - 300 µg/kg body weight (mouse, i.p.) (Rinehart et al., 1994; Sivonen and Jones, 1999). An exception is MCRR whose LD50 (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 IC50 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

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

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. LD50 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

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 tumour-promoting 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

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

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 LD50

of 2000 µg/kg bw for anatoxin-a (see 1.2.2.2) 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. LD50 of MCLR or Microcystis extracts appeared to be approximately a factor 30 - 170 lower than the oral LD50 (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 LD50s 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 LD50s 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.

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 [³H]-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/Oatp-mediated 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

The organotropism was additionally corroborated by various investigations

Im Dokument In vitro investigations on uptake and toxicity of cyanobacterial toxins (Seite 11-23)