Chapter

8
Cyanotoxins: An Emerging
Environmental Concern
RAKHI BAJPAI1, NAVEEN K. SHARMA2 AND ASHWANI K. RAI1,*
Department of Botany, Banaras Hindu University, Varanasi221 005, India
1

2
Department of Botany, PG College, Ghazipur233 001, India
*e-mail: akrai.bhu@gmail.com

ABSTRACT
1. INTRODUCTION
2. CYANOTOXINS
3. ENVIRONMENTAL FACTORS AFFECTING CYANOTOXIN PRODUCTION
4. ENVIRONMENTAL FATE OF CYANOTOXINS
5. ECOLOGICAL ROLE OF CYANOTOXINS
6. MICROCYSTINS
6.1. Biosynthesis of MCs
6.2. Techniques Used for Characterization and Detection of MCs
7. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
References
188 Advances in Life Sciences

ABSTRACT
Cyanobacteria (Blue-green algae) produce number of secondary metabolites, some are toxic
(known as cyanotoxins) to animals including human and other organisms, thus undesirable.
In recent years, due to eutrophication and ongoing climatic changes, incidences of freshwater
cyanobacterial blooms have increased substantially. Many of these surface bloom-forming
cyanobacteria are toxic. The chapter reviews the information available on various aspects of
cyanotoxins especially the factors affecting cyanotoxins production, environmental fate of
the cyanotoxins, and more importantly advantages provided to the emitter organisms by
cyanotoxins production (i.e., ecological role of cyanotoxins). Amongst different categories of
cyanotoxins, microcystins (MCs) are the most prevalent in various freshwater bodies across
the globe, therefore we have discussed the biosynthetic mechanism of microcystins (i.e.,
MC), as well as the techniques used for their identification, characterization and detection.

Keywords: Cyanotoxins, detection, environmental fate, health hazard, microcystins, mixed

polyketide synthases /non-ribosomal peptide synthetase (NRPS/PKS).

1. INTRODUCTION

In last 20 years, excessive pouring of nutrients (nutrient enrichment) and climatic changes have
resulted in exuberant growth of cyanobacteria (a condition termed as cyanobacteria bloom) in
freshwater bodies across the globe (Paerl & Huisman 2009). Cyanobacterial blooms develop
either at the surface (dense surface blooms are called scums) or in the metalimnion of water
bodies. Such blooms, referred to as CyanoHABs adversely affect the ecosystems and their
biota. Figure 1 presents the socio-ecological consequences of freshwater CyanoHABs (Van Dolah
2000; Havens 2007). CyanoHABs have far-reaching effect on species interactions, microbial
and macrophyte population, aquatic biota, human health, ecosystem integrity as well as industries
and economies (Carmichael 1997; Van Dolah 2000; Carmichael et al., 2001; Briand et al.,
2003).
Majority of the surface-bloom forming cyanobacteria produce toxins (cyanotoxins) and present
a potential risk to public health (Paerl et al., 2001; Codd et al., 2005). Climatic changes have
further laid to the geographical expansion of some toxic cyanobacteria. For instance,
Cylindrospermopsis raciborskii once reported from tropical waters has now moved to temperate
water bodies (Figueredo et al., 2007; Vidal and Kruk 2008).

2. CYANOTOXINS

Cyanobacteria produce variety of secondary metabolites (Table 1); some of them are toxic to
invertebrates, mammals and other aquatic organisms (Paerl et al., 2001; Codd et al., 2005).
However, secondary metabolites are not needed by the organisms in their primary metabolic
processes. Toxic cyanobacteria are widely distributed around the world and have been recorded
from every continent including Antarctica (Hitzfeld et al., 2000). Codd (1995) reported 50-75%
of the cyanobacterial blooms to be toxic. However, toxicity varies at species and intra-species
 Cyanotoxins: An Emerging Environmental Concern 189

Fig. 1. Summary of socio-ecological response and impacts associated with freshwater cyanobacterial blooms
(redrawn with permission from Havens 2007).

level, and with environmental conditions (Ouellette & Wilhelm 2003), since a toxic
cyanobacterium may or may not produce toxins (Ouellette & Wilhelm 2003). Likewise, it may
produce one or more than one toxic compounds (Codd et al., 2005). In freshwater habitats,
Microcystis is the most common toxic cyanobacterium. Other genera include Anabaena,
Oscillatoria, Nostoc, Anabaenopsis, Planktothrix, Aphanizomenon, Cylindrospermopsis,
Raphidiopsis, Lyngbya, Nodularia and Phormidium (Codd et al., 2005).
Cyanotoxins vary widely in their chemical nature and structure, toxic potency, mode of
action and organs affected (Falconer 2001; de Figueiredo et al., 2004), and are classified
accordingly (Sivonen & Jones 1999). The most widely used approach to categorize cyanotoxins
is based on the tissue/body part affected. Accordingly, cyanotoxins are: hepatotoxins (microcystins
and nodularins), neurotoxins (anatoxins and saxitoxins) and dermatotoxins (lipopolysaccrides)
190 Advances in Life Sciences
Table 1: Secondary metabolites produced by cyanobacteria.
Secondary Organism Bioactivity References
metabolites
Symplostatin 3 Symploca sp. Anticancer Luesch et al. 2002
Aplysiatoxin Lyngbya majuscula Anticancer Mynderse et al. 1977
Acutiphycin Oscillatoria acutissima Anticancer Barchi et al. 1984
Dragonamide C, D Lyngbya polychroa Anticancer Gunasekera et al. 2008
Cryptophycins Nostoc sp. Anticancer Moore et al. 1996
Arenastatin A Dysidea arenaria Anticancer Moore et al. 1996
Borophycin Nostoc linckia, Anticancer Hemscheidt et al. 1994
N. spongiaeforme
Homodolastatin 16 Lyngbya majuscula Anticancer Davies-Coleman et al. 2003
Curacin A Lyngbya majuscula Anticancer Simmons et al. 2005
Tjipanazoles Tolypothrix tjipanasensis Anticancer Bonjouklian et al. 1991
Pitipeptolides A, B Lyngbya majuscula Anticancer Luesch et al. 2001
Grassypeptolide Lyngbya majuscula Antiproliferative Kwan et al. 2008
Bauerines AC Dichotrix baueriana Anti-HSV-2 Larsen et al. 1994
Sulfolipids Lyngbya lagerhimii, Anti-HIV Gustafson et al. 1989
Phormidium tenue
Calcium spirulan Spirulina platensis Anti-HIV Hayashi et al. 1996
Cyanovirin Nostoc ellipsosporum Anti-HIV Dey et al. 2000
Venturamide A, B Oscillatoria sp. Antimalarial Linington et al. 2007
Dragomabin Lyngbya majuscula Antimalarial McPhail et al. 2007
Dragonamide A, B Lyngbya majuscula Antimalarial McPhail et al. 2007
Carmabin A,B Lyngbya majuscula Antimalarial, anticancer McPhail et al. 2007
Calothrixins A,B Calothrix sp. Antimalarial, anticancer Rickards et al. 1999
Dolastatins Lyngbya sp., Symploca sp. Antimalarial, anticancer Fennell et al. 2003
Nostocarboline Nostoc sp. Antimalarial, cholinesterase Barbaras et al. 2008
inhibitor
Symplocamide A Symploca sp. Antimalarial, anticancer Linington et al. 2008
Aeruginosins Microcystis, Nodularia, Serine proteases inhibitor Shin et al. 1997
Oscillatoria
Lyngbyastatin Lyngbya confervoides Serine protease inhibitor Taori et al. 2007
Anabaenopeptins Anabaena 90 Serine protease inhibitor Tonk et al. 2009
Cyanopeptolins Microcystis PCC 7806 Chymotrypsin inhibitor Bister et al. 2004
Radiosumin Plectonema radiosum Enzyme inhibitor Mooberry et al. 1995
Circinamide Anabaena circinalis Enzyme inhibitor Negri & Jones 1995
Agardhipeptin Oscillatoria agardhii Enzyme inhibitor Luukkainen et al. 1993
Norharmane Nodularia harveyana Enzyme inhibitor Volk 2006
Microviridin J Microcystis spp. Trypsin inhibitor Rohrlack et al. 2003
Bastadin Anabaena basta Antibiotic Miao et al. 1990
Bis-(c-butyrolactones) Anabena variabilis Antibiotic Ma & Led 2000
Nostocine A Nostoc spongiaeforme Antibiotic Hirata et al. 2003
Didehydromirabazole Scytonema mirabile Antibiotic Stewart et al. 1988
Tolyporphin Tolypothrix nodosa Antibiotic Prinsep et al. 1992b
Muscoride Nostoc muscorum Antibiotic Nagatsu et al. 1995
Kalkitoxin Lyngbya majuscula Block Na+ channels of nerve cells Wu et al. 2000
Antillatoxin Lyngbya majuscula Potent ichthyotoxin Berman et al. 1999
b-N-methylamino- Microcystis, Anabaena, lateral sclerosis/parkinsonism Cox et al. 2005
L alanine Nostoc and Planktothrix spp. -dementia complex
Spiroidesin Anabena spiroides Inhibits cell growth Kaya et al. 2002
 Cyanotoxins: An Emerging Environmental Concern 191

(Table 2). Lipopolysaccrides produced by most of the cyanobacteria have little health concern,
while hepatotoxins and neurotoxins have emerged as a major concern to the water supplying
agencies due to high exposure risk and lethality (Carmichael 1992; Fawell et al., 1994).

Table 2: Classification of cyanotoxins based on organs affected.

Cyanotoxin Mode of action Cyanobacteria Reference
Microcystins Hepatotoxic Microcystis spp., Planktothrix spp., Sivonen & Jones 1999; Cronberg et al.
Inhibits protein Anabaena, Nostoc, Synechocystis, 2003; Odebrecht et al. 2002; Ballot
phosphatase Cyanobium bacillare, Arthrospira et al. 2004; Gkelis et al. 2005; Baker
1/2A fusiformis, Limnothrix redekei, et al. 2001; Prinsep et al. 1992a;
Phormidium formosum, Lombardo et al. 2006; Bajpai et al.
Hapalosiphon hibernicus, 2009; Fiore et al. 2009.
Radiocystis feernandoi, Nostoc sp.
BHU001 Fischerella sp. strain
CENA161
Nodularin Hepatotoxic Nodularia spumigena Rinheart et al. 1988.
Inhibits protein
phosphatase
1/2A
Cylindrospermopsin Inhibits protein Cylindrospermopsis raciborskii, Ohtani et al. 1992; Harada et al., 1994;
synthesis Umezakia natans, Aphanizomenon Banker et al. 1997; Schembri et al.
ovalisporum, Aphanizomenon 2001; Li et al. 2001; Fastner et al.
flos-aquae, Rhaphidiopsis curvata, 2007; Spoof et al. 2006.
Anabaena lapponica, Anabaena
bergii
Anatoxin-a Neurotoxic Anabaena spp., Aphanizomenon Edwards et al. 1992; Sivonen et al.
Inhibits (A. flos-aquae, A. issatschenkoi), 1989; Park et al. 1993; Namikoshi
postsynaptic Cylindrospermum, Microcystis, et al. 2003; Wood et al. 2007.
depolarization Planktothrix spp., Raphidiopsis
mediterranea
Homoanatoxin-a Neurotoxic Oscillatoria formosa, Skulberg et al. 1992; Baker et al. 2001;
Inhibits Raphidiopsis mediterranea Namikoshi et al. 2003.
postsynaptic
depolarization
Anatoxin a-(s) Neurotoxic Anabaena flos-aquae, Carmichael & Gorham 1978; Henriksen
Inhibits acetyl A. lemmermannii et al. 1997.
cholinesterase
Saxitoxins (PSP) Neurotoxic Blocks Aphanizomenon, Anabaena, Humpage et al. 1994.
Sodium channel Lyngbya, Cylindrospermopsis
across the neuron spp.
LPS endotoxins Dermatotoxic All cyanobacteria McElhiney & Lawton 2005.
Skin and mucosa

In the natural freshwater bodies infested with cyanobacteria, microcystins (MCs) are the
most prevalent cyanotoxins. One of the best-documented incidences of cyanotoxin related human
causalities is from Caruaru (Brazil). In February 1996, 126 patients became seriously ill after
dialysis, of which 60 patients died in hospital. Later, it was found that water for dialysis came
from a local reservoir infested with heavy cyanobacterial bloom (Pouria et al., 1998; Jochimsen
et al., 1998). Analyses of the reservoir water revealed the presence of MCs -YR, -LR and AR.
MCs have been reported in a drinking water supplying reservoir in the Brazilian-Amazonia
region (Vieira et al., 2005). Nodularin has limited presence in cyanobacteria contaminated
freshwaters (Rinehart et al., 1988). Cylindrospermopsin (an alkaloid having a tricyclic guanidine
192 Advances in Life Sciences

linked to a hydroxymethyl uracil) (Ohtani et al., 1992), produced by Cylindrospermopsis

raciborskii) is another cyanotoxin reported from different freshwater bodies (Figueredo et al.,
2007).
In general, neurotoxins are less frequent in freshwaters hence, offer lesser exposure risk
than that of MCs (Fawell et al., 1994). Neurotoxins such as anatoxin-a, -a(s) and saxitoxins are
alkaloids, highly toxic to nerves. Anatoxin binds to the nicotinic receptor (has higher affinity
than that of acetylcholine) and acts as a postsynaptic depolarizing neuromuscular blocking agent
(Spivak et al., 1980; Carmichael 1997). Acute exposure of anatoxin causes death within minutes
to a few hours depending on the species, the amount of toxin ingested, and the quantity of food
in the stomach (Carmichael 1992). Edwards et al. (1992) reported that dog poisonings in Scotland
were due to the consumption of Oscillatoria containing anatoxin-a. Saxitoxin is a tricyclic alkaloid
that blocks neuronal transmission by binding to Na+ channels in nerve cells. Consequently,
sodium gradient is stopped leading to muscle paralysis and death (Kao & Nishiyama 1965).
Additional concern regarding the importance of cyanotoxins is reflected by their inclusion in
the US Environmental Protection Agency (USEPA) drinking water contaminant list and in major
reviews along with chemical warfare agents (Reemtsma 2003; Richardson & Ternes 2005).
Recent reports (2001-2009) on microcystin (MC) producing freshwater cyanobacterial bloom in
different parts of world are listed in Table 3.

Table 3. Freshwater microcystin producing cyanobacteria from different parts of the world (2001-2009).
Toxic Cyanobacteria
Planktothrix rubescens
Microcystis aeruginosa
Planktothrix rubescens
Anabaena sp., Microcystis sp.
Microcystis, Planktothrix, Anabaena
Microcystis aeruginosa
Microcystis aeruginosa,
Microcystis viridis
Strains LS703d, DVL1003c,
LS803, DVL1103b
Planktothrix agardhii
Anabaena circinalis,
Anabena spiroides
Nostoc sp. BHU001
Fischerella strain CENA161
Location & Country References
Lake Zrich, Switzerland Blom et al. 2001
Laguna de Bay, Philippines Baldia et al. 2003
Drinking water reservoir, Spain Barco et al. 2004
Mwanza Gulf (Lake Victoria), Tanzania Sekadende et al. 2005
Bangladesh freshwater ponds Welker et al. 2005; Ahmed 2009
King Talal Reservoir, Jordan Al-Jassabi & Khalil 2006
Romanian freshwater ponds Boaru et al. 2006
Drinking water reservoirs in Izaguirre et al. 2007
southern California
Viry-Chtillon lake, France Yprmian et al. 2007
Monjolinho Reservoir, Brazil Sotero-Santos et al. 2008
Agriculture Pond (BHU), Varanasi, India Bajpai et al. 2009
Capuava farm, So Paulo State, Brazil Fiore et al. 2009

3. ENVIRONMENTAL FACTORS AFFECTING CYANOTOXIN PRODUCTION

Light and temperature are the two main factors influencing cyanotoxins production. Utkilen and
Gjlme (1992) reported that under red and green light toxin production as well as toxicity
(toxin to protein ratio) of the cyanobacteria increases compared to that of white light. Toxicity
of the bloom also increases when light intensity decreases below 40 E m-2s-1 (Van der Westhuizen
& Eloff 1985; Watanabe & Oishi 1985; Utkilen & Gjlme 1992). Optimum temperature for
cyanotoxins production is 20-25C (Gorham et al., 1964; Watanabe & Oishi 1985). Utkilen and
Gjlme (1995) studied the effect of iron on toxin production by Microcystsis aeruginosa. Also,
 Cyanotoxins: An Emerging Environmental Concern 193

Jiang et al. (2008) used statistical approach to study the effect of different environmental factors
(light intensity, temperature and iron, etc.) on the growth and MCs production by Microcystsis
aeruginosa. Yan et al. (2004) found that iron up to 10 mg l-1 in the medium had little effect on
the growth of Microcystis aeruginosa DC-1, but enhances the production of MC-LR. The
intracellular MC content is related to N: P ratio of the medium. Maximal concentration of
intracellular MCs in different strains of Microcystis was recorded when initial N: P ratio in the
growth medium ranged between 458-664 for strain 205 and 237-753 for strain GL260735 (Vzie
et al., 2002). Level of cyanotoxins (extracellular) in a water body shows seasonal fluctuation
depending upon the physico-chemical factors of water bodies and the cyanobacterial species
dominating at particular time.

4. ENVIRONMENTAL FATE OF CYANOTOXINS

Cyanotoxins are either membrane-bound or occur free within the cells. They are released passively
in the medium with the aging and death of cyanobacterial population. During early growth
phase, cyanotoxins remain inside the cells and released in the environment at late-log growth
phase or after cell lyses (Chorus & Bartram 1999). Active release of toxins may also occur from
young growing cells (Pearson et al., 1990). Watanabe and Oishi (1983) investigated the toxicity
of a cultured strain of M. aeruginosa during different growth phases (lag, exponential and
stationary phases). They found maximal toxicity during late exponential or at stationary growth
phase. In another report, Watanabe et al. (1989) recorded maximal concentration of cell bound
toxin during late exponential growth phase. Release (concentration) of cyanotoxins in the medium
also increases with various water treatment practices.
In natural ecosystems, released cyanotoxins undergo photo- and bacterial degradation. In
addition, significant fraction of the released cyanotoxin becomes unavailable (for exposure) due
to adsorption over the soil surfaces depending upon environmental factors, soil property and
total organic content of the soil (Tsuji et al., 2006; Edwards et al., 2008). Degradation is generally
preceded by a lag period of about 9 to 10 days. For instance, MC-LR is stable in waters at high
pH and temperatures (upto 300C) (Wannemacher 1989), but readily biodegraded in ambient
waters with a half-life of about one week (Codd and Bell 1996). Cousins et al. (1996) reported
a half-life of less than a week for MC-LR. Figure 2 describes environmental fate of cyanotoxins
in natural ecosystems.

5. ECOLOGICAL ROLE OF CYANOTOXINS

Though substantial progress has been made on the toxicity of cyanotoxins, a little is known
about the benefit it provides to the producer (Paerl et al., 2001; Kaebernick & Neilan 2001;
Babica et al., 2006). Since, most of the organisms used in toxicity tests (invertebrates and
mammals) are neither natural enemies nor consumers of cyanobacteria (Pearl & Millie 1996),
ecological and physiological role of cyanotoxins still remains an enigma (Paerl et al., 2001;
Ouellette & Wilhelm 2003). In natural ecosystems, MCs are supposed to be involved in metal
ion chelation (Humble et al., 1997), intraspecific signaling (Dittmann et al., 2001), and protection
against predators such as zooplankton (Rohrlack et al., 2001) and allelopathic interactions against
194 Advances in Life Sciences

Fig. 2. Environmental fate of cyanotoxin in nature.

competitive photoautotrophic organisms (Pflugmacher 2002). Further, wide distribution and

diversity of microcystin isoforms itself indicates that microcystin production might be crucial
for natural adaptation of producer cyanobacteria in their natural environments.
Allelopathy refers to an inhibitory or stimulatory effect of plants and microorganisms on
other plant species or microorganisms through the release of organic compounds (Rice 1984).
 Cyanotoxins: An Emerging Environmental Concern 195

Such interactions occur in all aquatic habitats (Gross 2003) including phytoplankton communities
(Rengefors & Legrand 2001). Phytoplankton allelopathy alters succession and the pattern of
species dominance (Kearns & Hunter 2001; Prince et al., 2008). However, information related
to allelopathic role of cyanotoxins on associated bacteria (phycospheric biota) and other algae
(Paerl & Millie 1996; Sivonen & Jones 1999; Paerl et al., 2001) are meager.
Cyanotoxins have adverse effect on other aquatic biota (Zurawell et al., 2005). Use of
cyanobacteria infested water for irrigation is perceived as a threat to the yield and quality of
crop products (Bibo et al., 2008). Symptoms of MCs toxicity may be the result of direct action
of the toxic fraction, as well indirect involving oxidative damage induced by the toxin. Jrvenp
et al. (2007) found that exposure of MCs to broccoli (Brassica oleraceaGreenia Hg) seedlings
had mild (<10%) growth inhibitory effect, while mustard (Sinapis alba) seedlings remained
unaffected. MCs exposure inhibits superoxide dismutase and peroxidase activities in rape (Brassica
napus L.) and rice (Oryza sativa L.) seedlings resulting in oxidative damage to the plants (Chen
et al. 2004). Plants retain a substantial proportion of MCs present in the irrigation water, and
accumulate differentially in various parts, maximal being in root tissues (Jrvenp et al., 2007).
Growth inhibitory effect of MCs has been reported on Phaseolus vulgaris L., Solanum
tuberosum L. (McElhiney et al., 2001), Lepidium sativum L. (Gehringer et al., 2003), Brassica
napus L., Oryza sativa L. (Chen et al., 2004), Brassica oleracea var. italica, Sinapis alba
(Jrvenpa et al., 2007), Spinacia oleracea (Plugmacher et al., 2007), Pisum sativum, Lens
esculenta, Zea mays and Triticum durum (Saqrane et al., 2008). Uptake and translocation of
cyanotoxins into edible plant parts could expose consumers to medically relevant toxic
concentrations. The tolerable daily intake (TDI) of pure MC is 0.04 g Kg-1 body weight (Fawell
et al., 1994). Bioaccumulation of cyanotoxins in food chain and their effect on human health is
an important issue (Funari & Testai 2008).

6. MICROCYSTINS (MCs)

Bishop et al. (1959) were the first to recover MC from Microcystis aeruginosa strain NRC-1
(ss-17), and established its peptidal nature. The toxin(s) was named microcystin (MC) by Konst
et al. (1965). MCs are cyclic heptapeptides consisting of five common amino acids and two
variable L-amino acids (Fig. 3). The general structure of MCs is cyclo(-D-Ala-X-D-MeAsp-Y-
Adda-D-Glu-Mdha-), here, D-Ala and D-Glu are alanine and glutamic acid in D configuration,
respectively. D-MeAsp is 3-methylaspartic acid, Mdha is N-methyl-dehydroalanine, and Adda is
(2S, 3S, 8S, 9S)-3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid (Botes
et al., 1984; Rinehart et al., 1988; Tillett et al., 2000). X and Y refer to variable L-amino acids
at positions 2 and 4. Based on single letter code classification of amino acids, variables of MC
have been given different names depending upon the amino acids present at 2 and 4 positions
(variable positions) of the structure. For example, MC-LR (the most common cyanotoxin found
in water supplies around the world) contains amino acids leucine (L) and arginine (R) at the
position 2 and 4, respectively (Fig. 3) (Carmichael 1992). The unusual aromatic amino acid
Adda, a common constituent of all MCs is solely responsible for the toxicity of MCs (Sivonen
et al., 1992). Variations in the amino acids at other positions due to different degree of methylation
of Mdha and/or MeAsp as well as configuration of Adda also give rise to various MC variants
196 Advances in Life Sciences

Fig. 3. Structure of microcystin-LR (-D-Ala-L-Leu-D-MeAsp-L-Arg-Adda-D-Glu-Mdha-). Numbering of the various

amino acids are made as AA1 (D-Ala), AA2 (L-Leu), AA3 (D-MeAsp), AA4 (L-Arg), AA5 (Adda), AA6 (D-Glu)
and AA7 (Mdha).

(Table 4). In addition, there are many optical isomers of the microcystins. So far, more than 70
congeners of MC have been reported (Fastner et al., 2002; Jayaraj et al., 2006).

Table 4. Possible variations at respective amino acid positions (AA1- AA7) in microcystins. Amongst these, positions
2 and 4 have the highest number of variables. Abbreviations used are: Aba, amino-isobutyric acid; Adda,
(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid; ADM-Adda, O-acetyl- Adda; DM-
Adda, desmethyl-Adda; L-Glu(OMe), glutamate-methylester; Harg, homoarginine; H4Y, tetrahydro-tyrosine; Dha,
dehydroalanine; Dhb, dehydrobutyric acid; Mdha, N-methyl-dehydroalanine; Me, methyl; MeLan, N-methyl-lanthionine.
AA1 AA2 AA3 AA4 AA5 AA6 AA7
D-Ala L-Leu D-MeAsp L-Ala Adda D-Glu Mdha
D-Ser L-Ala D-Asp Aba DM-Adda D-Glu(OMe) Dha
D-Leu L-Tyr L-Leu (6Z)Adda L-Ser
L-Glu L-Arg ADM-Adda L-MeSer
L-Val L-Glu Dhb
L-Glu(OMe) L-Glu(OMe) MeLan
L-Arg L-Phe
L-Phe L-Tyr
L-Met(O) L-Har
L-(H)Phe L-Trp
L-(H)Tyr L-Met(O)
L-Trp L-(H)Arg
L-(H)Arg L-Glu
L-(H)Ile
L-H4Y
 Cyanotoxins: An Emerging Environmental Concern 197

During bloom, different variants of MCs may be produced causing repeated but varied
poisoning in animals visiting the water bodies. The levels and relative proportions of MC variants
are regulated by external growth stimuli (Rapala et al. 1997). World Health Organization (WHO)
has set a threshold limit (1 g l-1) for MC-LR in drinking waters. The International Agency for
Research on Cancer (IARC) has classified MC-LR as a possible human carcinogen (group 2B)
(IARC 2006; Grosse et al., 2006). Chronic consumption of MCs present in tap waters (at lower
doses) could be a substantial risk factor for liver and colorectal cancer (Hernndez et al., 2009).
MCs have been implicated in numerous cases of liver cancer (Nishiwaki-Matsushima et al.,
1992; Codd et al., 2005). They promote tumor formation in cultured cell lines. Transport of
MCs into the hepatocytes occurs via bile acid carriers (Eriksson et al., 1990). Inside hepatocytes,
MCs induce alteration in the microfilaments of the cells leading to their aggregation near the
center of the cells. Consequently, cellular support is lost and hepatocytes become round causing
destruction of the sinusoid endothelium. At biochemical level, MCs inhibit protein phosphatases
1 and 2A (differ in substrate specificity) in mammals and higher plants (Honkanen et al., 1990;
MacKintosh et al., 1990). Protein kinases and protein phosphatases regulate cellular signaling
through phosphorylation and dephosphorylation of the proteins. Phosphorylated proteins play a
significant role in expression of the genes involved in cell growth (Nishiwaki-Matsushima et al.,
1992). Protein phosphatases control a range of cellular functions including glycogen metabolism,
phosphorylations of cytoskeleton, RNA splicing, muscle relaxation, cell-cycle progression to
telomerase activity and apoptosis (Ohta et al., 1992; Terrak et al., 2004). They specifically
hydrolyse serine/threonine phosphatase (serine/threonine phosphoeasters) and protein tyrosine
phosphatase (phosphotyrosine) (Denu et al., 1996). Serine/threonine protein phosphatases (PP)
1, 2A, 4, 5, and 7 are evolutionarily conserved, and inhibited by toxins of marine dinoflagellates
(okadaic acid), terrestrial fungi (calyculin A), cyanobacteria (microcystins, nodularin), soil bacteria
(tautomycin) and venomous insects (cantharidin). Krakstad et al. (2006) reported that the PP-
inhibiting toxins induce morphological alterations compatible with apoptosis in different cell
types.
MCs inhibit protein phosphatase specifically type-1 (PP1) and type-2 (PP2A) (members of
protein serine/threonine phosphatase family) in liver cells causing rapid reorganization of all
three major cytoskeleton components, microfilaments, microtubules and intermediate filaments
(MacKintosh et al., 1990; Toivola & Eriksson 1999). Imanishi and Harada (2004) extracted
MC-binding protein phosphatases (PP1, PP2A, and PP4) from mice liver treated with MC using
two dimensional gel electrophoresis and MALDI-TOF MS.

6.1 Biosynthesis of MCs

MCs are synthesized via a mixed polyketide synthases (PKSs), non-ribosomal peptide synthetase
(NRPSs) and hybrid synthetases (NRPS-PKS and PKS-NRPS) through thio-template mechanism
(Moore et al., 1991; Dittmann et al., 1997, 2001). Though polyketides and nonribosomal peptides
appear structurally unrelated, they are assembled in similar manner. NRPS and PKS are large
multifunctional complexes organized in modules. Each module is responsible for one cycle of
polyketide or polypeptide chain elongation. The modules are subdivided into domains, which
are enzymic units catalyzing the individual steps of polyketide and polypeptide synthesis. There
198 Advances in Life Sciences

are three domains, adenylation-(A)-domain, peptidyl carrier protein (PCP), and condensation-
(C)-domain for the biosynthesis of the peptide backbone. The adenylation-(A)-domain is required
for substrate recognition and activation. It controls the entry of substrates into non-ribosomal
peptide synthesis by selection of amino acids, and simultaneously their activation as aminoacyl
adenylates at the expense of ATP. The peptidyl carrier protein (PCP) transports activated amino
acids to the respective catalytic centers (also known as thiolation-(T)-domain). The condensation-
(C)-domain helps in the formation of peptide bond (Schwarzer et al., 2003). Besides, there is
N-methylation (methylation of amino groups) and epimerization (convert L-amino acids to
D-isomers) domains. Order of these modules together with the number and type of catalytic
domains determines the structure of the resulting polyketide or peptide product (Tillett et al.,
2000). There is a correlation between the sequence of modules and the biosynthetic steps.
Biosynthesis of non-ribosomal peptides is a very complex process. Amino acids constituting
the structure of the peptide are incorporated sequentially, through a series of partial and repetitive
enzymatic reactions. The final peptidal product is liberated through a thioesterase activity catalyzed
by the thioestrase domain. Prior to biosynthesis, NRPSs which are in inactive (apo) state get
converted to holo- form by a specific post-translational reaction. Which includes transfer (catalyzed
by enzyme phosphopanthotenyl transferase) of the cofactor 4-phosphopanthetein (PAN) to the
serine residue (present in the conserved motif, LGGDSL) of PCP domain. Primary precursors
required for MC biosynthesis are phenylacetate, malonyl-CoA, S-Adenosyl-L-methionine (SAM),
glutamate, serine, alanine, leucine, D-methyl-isoaspartate and arginine (Tillett et al., 2000).
A total of 48 sequentially catalyzed reactions are involved in MCs synthesis, out of which
45 are assigned to catalytic domains organized within six large multienzyme synthases/synthetases
(McyA-E and G) (Kaebernick et al., 2002). These reactions are involved in incorporation of
primary precursor molecules. The remaining four-monofunctional proteins are supposed to be
involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI) and localization
(McyH). The later (McyH) shows high identity to ABC transporter genes. The unusual polyketide
amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound
intermediate, and not released during the process (Tillet et al., 2000) (Fig. 4). Kaebernick and
Neilan (2001) and Dittmann and Brner (2005) have provided detailed information about the
MCs biosynthesis.
Tillett et al. (2000) identified MC biosynthetic (mcy) gene cluster of 55kb located on the
chromosomes in Microcystis strain PCC 7806. It is composed of 10 bidirectionally (oppositely)
transcribed open reading frames (ORFs) arranged in two operons (mcyA-C and mcyD-J). These
ORFs encode for 10 proteins, six being multifunctional enzymes composed of PKS and NRPS
domains. The larger of the two operons (mcyD-J) encodes the PKS-NRPS modules catalyzing
the formation of the pentaketide-derived b-aminoacids Adda and its linkage to D-glutamate. The
smaller (mcyA-C) encodes NRPS modules for the extension of this dipeptidyl intermediate to
the heptapeptidyl step and subsequent peptide cyclization (Tillett et al., 2000). ORFs are flanked
on both sides by genes not involved in MC biosynthesis. The mcy gene cluster of Anabaena
(Rouhiainen et al., 2004), Microcystis (Tillett et al., 2000), Planktothrix (Christiansen et al.,
2003), and of nodularin in Nodularia (Moffitt & Neilan 2001, 2004) is shown in Fig. 5. A
comparison of mcy gene clusters in different cyanobacteria indicates that majority of the gene
are in different order, and not all the genes are present in each cyanobacterium. This necessitates
characterization of mcy gene clusters of individual cyanobacterium.
Fig. 4. Microcystins biosynthetic model. White rectangles represent peptide synthetase domains (NRPS); black rectangles thiolation motif of NRPS,
and grey rectangles indicate polyketide synthase domain (PKS). Number indicates the order in which NRPS incorporates individual microcystin
moieties. Solid arrows indicate PKS and broken arrows NRPS catalyzed steps. A = adenylation, C = condensation, Ep = epimerization and NMT =
N-methyltransferase (after Kaebernick & Neilan 2001; Dittmann & Brner 2005).
Cyanotoxins: An Emerging Environmental Concern 199
200 Advances in Life Sciences

Fig. 5. Distribution of mcy gene cluster in various cyanobacterial genera. Arrows indicate the transcriptional
start sites from the putative promoter regions (after Tillett et al., 2000, Christiansen et al., 2003, Rouhiainen
et al., 2004; redrawn from Dittmann & Brner 2005).

6.2 Techniques Used for Characterization and Detection of MCs

Various techniques (i.e., biochemical, analytical and molecular) are in use to characterize and
detect MCs in water samples (Fig. 6). Amongst these, mouse bioassay test is one of the oldest
approaches used to characterize cyanotoxins (Porfirio et al., 1999; Chorus & Bartram 1999).
This is useful in qualitative analysis and estimation of the lethal doses of the toxins, but fails to
detect the toxins at low level (Lambert et al., 1994). Growing concern for animal ethics has
further reduced the feasibility of this assay. An and Carmichael (1994) developed protein
phosphatase inhibition assay for MCs analysis.
Immunoassay techniques due to their high sensitivity, specificity and operational simplicity
are widely used for the characterization of MCs. Enzyme linked immunosorbent assay (ELISA)
can detect low concentration of MCs (1 g l-1) and requires minimum sample processing
(McElhiney & Lawton 2005). ELISA kits have been developed using both polyclonal (Metcalf
et al., 2000) and monoclonal antibodies (Nagata et al., 1995; Zeck et al., 2001). However,
water samples often contain more than one toxin that may render ELISA unsuitable. Recently,
Lawton et al. (2009) have developed immunostrips that can be used for rapid detection of MCs
and nodularin in natural samples.
 Cyanotoxins: An Emerging Environmental Concern 201

Fig. 6. Techniques commonly used for the detection of microcystins in natural sample.

Amongst chromatographic techniques, thin layer chromatography (TLC) is the easiest

technique used for the characterization of MCs (Pelander et al., 1996). However, it is unspecific
and less sensitive. Use of high performance liquid chromatography (HPLC) for the identification
and quantification of MCs has increased greatly. Lawton et al. (1994) developed a reverse-
phase HPLC method to analyze MCs and nodularin in both raw and treated water. This method
involves filtration to separate cyanobacterial cells from waters allowing intracellular and
extracellular (in medium) level of toxins to be assessed. Filtered water is subjected to enrichment
using a C18 solid-phase extraction cartridge, followed by identification and determination by
photodiode array (Lawton & Edwards 2001). This technique uses both gradient elution and
isocratic mobile phases for the separation of MC variants. However, McElhiney and Lawton
(2005) recommend using the gradient to resolve wide range of toxin variants. In majority cases,
toxins are separated on C18 silica column using a gradient of water and acetonitrile as mobile
phase. The chromatographic efficiency of mobile phase can be improved considerably by addition
of perfluorinated alkyl carboxylic acids such as trifluoroacetic acid 0.05% (TFA), which acts as
an ion-pairing agent (Lawton et al., 1994). However, ability of this technique to distinguish MC
202 Advances in Life Sciences

variants is limited as most variants have a similar absorption profile between 200 and 300 nm
(Harada et al., 1999).
To get an accurate identification of MCs, HPLC is followed by mass spectrometry (LC-MS)
(Harada et al., 2004). Different configurations of this approach such as fast atom bombardment
(FAB) (Kondo et al., 1995) and electrospray ionization (ESI-MS) have been developed (Barco
et al., 2002; Spoof et al., 2003). Zhang et al. (2004) used LCMSMS with electrospray
ionization to determine MC-LR in surface waters. According to Harada et al. (2004), LC-ESI-
MS provides information on retention behavior, molecular weight and ion intensity of the MCs,
thus making easy to distinguish MCs from other peptides.
Due to the unavailability of analytical standards, MC variants are identified with reference
to purified MC-LR, and concentrations are expressed in terms of MC-LR equivalence. MCs are
also identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS). The technique requires a small amount of sample without separation or
purification (Welker et al. 2002). Erhard et al. (1997) used MALDI-TOF MS for identification
of secondary metabolites with intact cyanobacterial cells. Resulting mass signals of MC variants
are further characterized by postsource-decay fragmentation, and comparison of observed fragment
spectra with theoretical ones or with those of pure reference compounds (Welker et al., 2004).
In general, MALDI-TOF MS is used for the identification of peptides; however, this can also be
used for the identification of alkaloids (Araoz 2008). In MALDI-TOF MS, the mass fragmentation
pattern of a particular analyte is distinctive, and may vary according to the ionization mode
used for mass spectrometry and to the charge state of the molecule (Antoine et al., 2006;
Welker et al., 2006). Addition of 200 M zinc sulfate heptahydrate (ZnSO4.7H2O) to the sample
prior to spotting, enhances the detection of the protonated molecule, while suppresses competing
adducts (Howard & Boyer 2007). For complex samples, this technique provides a highly simplified
spectrum with enhanced quantitative analysis. Further, MALDI-TOF-MS needs no sample
purification, and can be performed with lyophilized cell or colonies. Recently, the high-throughout
technique such as DNA microarray is being used for detection and differentiation of toxic
cyanobacterial genotypes (Pearson & Neilen 2008).
PCR-based amplification of different domains of mcy gene clusters has greatly enhances the
molecular differentiation between toxic and non-toxic strains (see Pearson & Neilen 2008).
However, this approach only indicates about the potentiality of a cyanobacterium to produce
toxins. Studies revealed that a cyanobacterium containing mcy gene may not necessarily produce
toxins. It depends upon differential expression of genes in response to environmental factors.

7. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Molecular techniques have revolutionized our understanding of microbial systematics and ecology.
The discovery of mcy gene clusters in Microcystis, Planktothrix and Anabaena has opened new
ways to rapid and accurate characterization of toxic cyanobacteria. However, cyanobacteria with
mcy gene may not necessarily synthesize cyanotoxins depending upon the environmental factors.
Therefore, to minimize the risks associated to cyanotoxins exposure, there is a need to monitor
toxin producing cyanobacterial species, and environmental factors responsible for the production
of cyanotoxins. Further, role of the cyanotoxins may also be understood by the use of mutant
 Cyanotoxins: An Emerging Environmental Concern 203

cyanobacterial strains. In such mutants, gene(s) for a known cyanopeptide is knocked out, but
others are allowed to transcribe. This would reveal that internal cell regulation compensates for
the missing peptide in some way, which would be a step towards understanding peptide functions
for the cell.

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