1.2 Cyanobacterial Toxins
1.2.3 Other Cyanobacterial Toxins
The non-protein amino acid β-N-methylamino-L-alanine (BMAA; Fig. 1.6) has initially been isolated from extracts of cycad seeds (Cycas circinalis) from Guam, an island in the western Pacific Ocean (Vega and Bell, 1967; Vega et al., 1968).
Recently Cox et al. (Cox et al., 2003; Cox et al., 2005) found that BMAA is of cyanobacterial origin and accumulates in cycad seeds as a result of a symbiosis between the cycad coralloid roots and a BMAA producing Nostoc species. The authors furthermore reported that BMAA increasingly accumulates in higher trophic levels, i.e. flying foxes (Pteropus mariannus) and the Chamorro, the indigenous population of Guam that consume both cycad seeds and flying foxes as part of their traditional diet.
Fig. 1.6: Structure of β-N-Methylamino-L-Alanine (BMAA).
BMAA was found to induce a neurological disorder (i.e. corticomoto-neuronal dysfunction, chromatolytic and degenerative changes of motor neurons and symptoms similar to Parkinson’s) in macaques following oral administration (Spencer et al., 1987a). Therefore, it has been hypothesized to be the causative agent for the increased incidence of amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) among the Chamorro, as well as the indigenous Auyu of Irian Jaya, Indonesia and the Japanese residents of the Kii peninsula of Honshu island, where cycad seeds are also part of the traditional diet or used in topical medicine (Spencer et al., 1987b; Spencer et al., 1987c). ALS/PDC is a severe tauopathy that shares similarities to amyotrophic lateral sclerosis, Parkinson’s disease and Alzheimer’s (Steele, 2005).
Indeed, BMAA has been shown to be neuro- and excitotoxic on cultured mouse cortical neurons by acting as an agonist of glutamate receptors at relatively high concentrations and in dependence upon the presence of physiological concentrations of bicarbonate ions (Weiss et al., 1989a; Weiss et al., 1989b).
Bicarbonate serves as a cofactor for the reversible reaction of BMAA with dissolved carbon dioxide to β-carbamate that mimics the effect of glutamate (Myers and Nelson, 1990). Moreover, in rat brain cells BMAA dose-dependently elevates intracellular calcium levels in the presence of bicarbonate ions (Brownson et al., 2002), an effect that is known to potentially induce cell death and neurodiseases.
As suggested by the aforementioned macaque study (Spencer et al., 1987a) BMAA reaches the brain after oral application. In fact, 80 - 100% of the p.o.
administered dose becomes bioavailable as shown in another macaque experiment (Duncan et al., 1992). Smith et al. (Smith et al., 1992) identified the cerebrovascular large neutral amino acid carrier as being responsible for the uptake of BMAA across the blood-brain-barrier in rats.
Furthermore, BMAA not only exists as a free amino acid, but it also occurs in a protein-bound form that usually exceeds the former 10- to 240-fold (Murch et al., 2004b). In addition, Murch et al. concluded that protein-bound BMAA may form an endogenous reservoir from which free BMAA is slowly released by protein metabolism. They further hypthesized that this slow release might cause continuous damage to the brain providing an explanation for the long latency
Chapter I General Introduction
period of ALS/PDC from years to decades (Spencer et al., 1991a; Kisby et al., 1992).
1.2.3.2 Lipopolysaccharides
A common component of the outer cell membrane of gram-negative prokaryotes, including cyanobacteria, are lipopolysaccharides (LPS). As their name indicates LPS consist of carbohydrates (core polysaccharides and an outer polysaccharide chain) and lipids (lipid A) whose composition is very variable among bacteria in general, but also among cyanobacteria (Sivonen and Jones, 1999; Briand et al., 2003; Wiegand and Pflugmacher, 2005).
In contrast to the aforementioned cyanotoxins, LPS are endotoxins that may elicit irritant, pyrogenic, allergic and toxic effects predominantly by the fatty acid component (Weckesser et al., 1979; Kuiper-Goodman et al., 1999; Sivonen and Jones, 1999; Briand et al., 2003). However, cyanobacterial LPS seem to be less toxic compared to LPS from pathogenic gram-negative bacteria, e.g.
Salmonella (Kuiper-Goodman et al., 1999; Briand et al., 2003; Wiegand and Pflugmacher, 2005).
2 Objectives
The primary molecular mechanism underlying the toxicity of MCs and NODs (i.e. PP inhibtion) has been extensively investigated and is well comprehended.
As mentioned in the previous chapter, the Adda moiety is of crucial importance for the inhibition of PPs and thus toxicity. However, isolated Adda was demonstrated to lack inhibitory activity on PP2A (Harada et al., 2004). In order to complete this finding and to exclude potential differences in the effects of Adda on PP1 and 2A, colorimetric PP inhibition assays were conducted in this study with both phosphatases (chapter III).
Although numerous studies focused on the organotropism of MCs, only little information exists on the transporters that mediate the cellular uptake and excretion of MCs, as well as their role in congener-specific toxicity. The latter appeared to significantly vary among some congeners in vivo despite similar PP inhibitory capacities (e.g. MCLR and MCRR). Therefore, the role of human liver OATPs in congener-specific in vitro toxicity of four different MCs (MCLR (leucine, arginine), MCRR (arginine, arginine), MCLW (leucine, tryptophan) and MCLF (leucine, phenylalanine)) was examined using stably OATP1B1- and OATP1B3-transfected HEK293 cells and primary human hepatocytes (chapter IV). As a prerequisite for the comparison of both, the congeners and the different cell types, the toxicodynamics of these four MC congeners, i.e. their inhibitory capacity on recombinant and endogenous serine/threonine-specific PPs, were assessed.
Furthermore, the risk emanating from exposure to CYN predominantly via drinking water is increasing, along with the abundance of its producers.
Especially Cylindrospermopsis raciborskii, has been reported to be invasive and to spread from tropical and subtropical regions into more temperate climate (Padisak, 1997; Fastner et al., 2003; Neilan et al., 2003; Falconer and Humpage, 2006). Despite intensive research the molecular mechanisms of CYN are not completely understood and human toxicity has barely been adressed. Most in vitro studies on the toxicity of CYN were carried out on
Chapter II Objectives
permanent cell lines and primary hepatocytes of mice and rats (Runnegar et al., 1995d; Shaw et al., 2000; Chong et al., 2002; Froscio et al., 2003; Humpage et al., 2005). In addition, the findings of Chong et al. (Chong et al., 2002) suggest the involvement of the bile acid transport system in facilitating the uptake of CYN. Thus, a further scope of this study was to determine the cytotoxicity of CYN along with the involvement of liver OATPs in primary human hepatocytes and OATP1B1- and OATP1B3-expressing HEK293 cells (chapter V).
Contact to cyanotoxins may occur via several routes of exposure as summarized in the previous chapter. Of major concern are contaminations of food and drinking water, whereas the latter especially poses a threat for poorer countries where water treatment is limited or absent (Falconer, 1993; Dietrich and Hoeger, 2005; Falconer, 2005a; Falconer and Humpage, 2005b). However, in industrialized countries cyanobacterial dietary supplements (blue-green algae supplements (BGAS)) that are consumed for their putative beneficial health effects (i.e. increased alertness and energy, “detoxification”, efficacy against various viral infections, cancer and mental disorders like depression or attention-deficit disorders) represent an exceptional source for cyanotoxin exposure, in particular MCs (Gilroy et al., 2000; Kuiper-Goodman et al., 2000;
Lawrence et al., 2001; Dietrich and Hoeger, 2005; Saker et al., 2005).
Furthermore, as BMAA was not only detected in brain tissues from Chamorros who died from ALS/PDC, but also in brain tissues from Alzheimer patients from Canada (Cox et al., 2003; Murch et al., 2004a), which raises the question about the corresponding source of BMAA. According to the findings of Cox et al. (Cox et al., 2005) BMAA may be produced by all known groups of cyanobacteria.
Therefore, besides assessing the health risk of a potential contamination of different BGAS samples with MCs using different analytical methods, a further aim was to analyze these supplements for contamination with BMAA (chapter VI).