Persistence and Decomposition of Hepatotoxic Microcystins Produced by Cyanobacteria in Natural Environment

J. TOXIC0L.-TOXIN REVIEWS, 17(3), 385-403 (1998)

Persistence and Decomposition of Hepatotoxic Microcystins Produced by Cyanobacteria in Natural

Environment

Ken-ichi Haradal and Kiyomi Tsuji2

1Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan 2Kanagawa Prefectural Public Health Laboratory, Nakaecho 52, Asahi-ku, Yokohama

241 Japan

ABSTRACT

Microcystins, the cyclic heptapeptide toxins produced by cyanobacteria such as

Microcystis, show tumor-promoting activity through inhibition of protein phosphatases 1

and 2A. They potentially threaten human health and are increasing the worldwide

interest in the health risk associated with cyanobacterial toxins. Microcystins are

normally considered to be. confined within cyanobacterial cells and to enter into the

surrounding water after lysis and cell death under field conditions. Five pathways may

be considered to contribute to natural routes of detoxification of the microcystins: (1)

dilution, (2) adsorption, (3) thermal decomposition aided by temperature and pH, (4)

photolysis and (5) biological degradation. In this review, we describe the persistence

and decomposition of microcystins under the conditions mentioned above and discuss the

fate of the toxins in the natural environment.

INTRODUCTION

Toxic blooms of cyanobacteria occur in eutrophic lakes, ponds and rivers all over

Cyanobacteria produce acute toxins such as hepatotoxic peptides, the world.

385

Copyright 6 1998 by Marcel Dekker. Inc. www,dekker.com

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386 HARADA AND TSUJI

microcystins and nodularins, and neurotoxic alkaloids, anatoxin-a, anatoxin-a(s) and

aphantoxins. Microcystis, OsciuarOria, Anabaem, Aphanizomenon and Nodularia

produce these toxins and have been responsible for the death of wild and domestic

animals consuming water contaminated with bloom. Recently a new hepatotoxin.

cylindrospermopsin was isolated from Cylindrospennopsis and Umezakia (1). The

principal species under investigation, Microcystis aeruginosa, has been frequently cited in

animal poisoning incidents and produces strongly hepatotoxic cyclic heptapeptides called

microcystins. The toxins consist of a common moiety composed of seven amino acids.

Approximately Mother components have been isolated so far. The toxins also inhibit

protein phosphatases 1 and 2A in a manner similar to okadaic acid and have a tumor-

promoting activity in rat liver. Microcystins as liver tumor promoters would threaten

human health (2).

TIME (April 22, 19%) reported an article in which 126 patients who underwent

hemodialysis at the ludney disease institute in a Brazilian town between February 13 and

16, 19%, fell ill with symptoms raging from nausea and blurred vision to convulsions

and internal hemorrhaging(3). T h s was caused by microcystin-LR, which was detected

in the water and the filters of the dialysis rnachmes. To date, 55 persons have died, and

this is without a doubt the first confirmation of human deaths from microcystins.

Microcystins are cyclic heptapeptides and are chemically very stable. Our

preliminary experiment on stability of the toxins showed that the half-life was estimated to

be three weeks in solution even at pH 1 and 40°C. To degrade them completely, i t is

necessary that they be treated under strong acidic conditions such as 6N hydrochloric acid

and TFA (trifluoroacetic acid) under reflux. The toxins are also resistant to enzymatic

hydrolysis by the usual enzymes, such as trypsin.

Microcystins are normally considered to be confined within cyanobacterial cells and

to enter into the surrounding water after lysis and cell death under field conditions. As

will be shown later, however, the amount of microcystins detected in lake water was, at

most, a few pgll, and the amount was much less than that estimated in the cells. To

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HEPATOTOXIC MICROCYSTINS 387

assess health implications, it is very important to pursue microcystins under field

conditions. Few systematic detoxification studies on microcystins have been

conducted. Five pathways may be considered to contribute to natural routes of the

detoxification of microcystins: ( 1) dilution, (2) adsorption, (3) thermal decomposition

aided by temperature and pH, (4) photolysis and (5) biological degradation.

In this review, we describe the persistence and decomposition of microcystins

under the conditions mentioned above and discuss the fate of the toxins in the natural

environment.

MICROCY STINS

Structures

Botes et al. isolated several microcystins from a South African Microcystis

mginosa strain (4) and first determined the structure of one of them (designated as

cyanoginosin) in 1984 (5). In the next year, the same group published structures of the

remaining toxins, as shown in Figure 1 (6). The structures of these microcystins were

uniformly determined to be monocyclic and composed of the D-amino acid, alanine (Ala);

f3-linked erythro-f3-methylaspartic acid (f3-Me-Asp); y-linked glutamic acid (Glu); two

variable L-amino acids with combinations known to include, for example, leucine and

alanine (LA), leucine and arginine (LR), tyrosine and arginine (YR), tyrosine and alanine

(YA). and tyrosine and methionine (YM); and two unusual amino acids, N-

methyldehydroalanine (Mdha) and 3-amino-9-methoxy-2,6,8-tnmethyl- 10-phenyldeca-

4(E),qE)-dienoic acid (Adda). The stereochemistry of Adda has been determined and

assigned as 2s. 3S, 8S, 9s , thus completing the absolute structures of microcystins (7).

Once the basic structure was determined, many reports on microcystins quickly

followed. More than 50 microcystins have been isolated to date and the structural

variations in microcystins are summarized by Rinehart et al. (8). One of the

characteristic structural features in microcystins is the presence of a f3-amino acid, Adda;

thls amino acid is also contained in nodulanns (7). A geometrical isomer at C-7 in the

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388 HARADA

6

AND TSUJI

X Y micmcystin-LA Leu Ala

-LR Leu A r g -YR Ty Arg -YA Ty Ah -YM Ty Met

Fig. 1 Structures of five microcystins proposed by Botes et al.

Adda moiety having qE), 6(2) configuration (abbreviated as 6(Z)-Adda microcystin)

shows no toxicity, therefore, the geometry of 4(E), 6(E) is considered to be essential for

the biological activity (9-1 1). This amino acid increases the hydrophobicity of the whole

microcystin molecule. Microcystins possess another unusual unsaturated amino acid,

Mdha, which serves as a Michael addition acceptor. Indeed, microcystins react smoothly

with glutathione and cysteine to yield their adducts (12) Very recently, it was found that

microcystin is bonded covalently with the cysteine moiety of protein phosphatases

(13,14).

Biological activity

By the consumption of water contaminated with microcystins, many kinds of

animals (cattle, sheep, hogs, birds, fish, dogs and horses) have been affected (15).

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Purified microcystins cause death in laboratory mice and rats within 1-3 hr. The liver is

consistently the most severely affected organ, showing extensive hemorrhagic necrosis

and disruption of the sinusoids. This vascular congestion of the liver leads to a

doubling of liver weight, but other organs seem normal. When microcystin-LR was

injected intraperitoneally into mice, the observed LDm values varied from 50 to 1 0 0

Fgg/kg (16). The chronic administration of Microcystis extract at low concentration in

the drinking water of mice resulted in increased mortality, particularly in male mice,

together with chronic active liver injury (17).

The findmg that microcystin activated phosphorylase A preceded studies which

show that microcystins are potent inhibitors of protein phosphatases 1 (PPl) and 2A

(PP2A) (18-21). These observations are important since inhibition of PP1 and PP2A

indicates that microcystins are likely to be tumor promoters. Because microcystins are

preferentially taken up by hepatocytes, it is expected that the main health threat as a tumor

would be in liver tumor promotion. Nishiwalu-Matsushima etal. have shown a two-

stage tumor promotion study which demonstrates tumor promotion in rat liver by

microcystin-LR (22). These results suggest that microcystins are a potential health

threat in drinking water supplies where cyanobacterial blooms are dominant for extended

periods of time.

CURRENT SITUATlON OF MICROCYSTIN LEVEL IN JAPANESE LAKES

Lakes Sagami and Tsukui are located near Tokyo and are two of the

representative eutrophic lakes in Japan. We have continued to survey the microcystin

level in Lakes Sagami and Tsukui for the past five years, which were created when dams

were constructed across the Sagami Rver in 1945 and 1%5, respectively. They are

water reservoirs serving a population of approx. 5,000,000, and a means of electric

power production and are also important recreational areas. However, cyanobacterial

blooms have frequently occurred in these lakes due to eutrophication since 1979. In

order to prevent the outbreak of cyanobacterial blooms, an aeration system has been

introduced in Lakes Sagami and Tsukui since 1992 and 1994, respectively.

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Raw lake waters from both lakes were separated into cyanobacterial cells and

cell- free water and were analyzed for microcystins over four years. Our established

method using HPLC allowed a precise qualitative and quantitative analysis of

microcystins in both fractions from lake water at a 0.02 pg/l level (23). Table 1 shows

the dominant cyanobacteria species observed, the concentration of microcystins and

chlorophyll a in the lake water together with the water temperature.

The water temperature of Lake Tsukui was generally hgher than that of Lake

Sagami on the same day. In Lake Sagami, the frequency of AnabacM was higher than

that in Lake Tsukui. Microcystins were found to be present at 0.13-3.10 pg/ l in the

cells, but none was detected in cell-free water over the four years of the study.

Microcystins in the cell-free water of Lake Tsukui were found at 0.02-3.80 pg / l , while

they were 0.04-482 pgll in cell samples. Although microcystins-RR and -LR were

mainly found in these lakes, a small amount of -YR was occasionally detected in the

samples. The contents of microcystins-RR, -YR and -LR in Microcystis lyophilized

cells (0.1 g) from Lakes Sagami andTsukui were 0.4 -132 pg, 42-12.3 pg and c0.2-

58 p g , respectively. Cyanobacterial blooms were observed at all sampling sites, with

particularly high concentrations of microcystins and chlorophyll a. The total content of

microcystins was estimated to be approx. 500 pg/l in this lake water. Microcystins in

the cells showed a similar pattern for this 4-year period. Recently. three countries,

Australia, Canada and Great Britain, are moving to establish maximum acceptable levels

for microcystins in drinking water supplies (15). It is recommended that the guideline

level be 1.0 pg microcystinslnodularinsll. Although there were many cases where

microcystin concentrations in cells exceeded the guideline, there was only one case in

water samples (24).

The microcystin content in cell-free water was approx. 5% of the total amount of

microcystins, which seemed to be a lower percentage than that estimated in the entire

water sample. In order to observe the release of microcystins into the surrounding

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TABL

E 1.

C

once

ntra

tion

of M

icro

cyst

ins

Con

tain

ed in

Lake

Wat

er, J

apan

* 2

Lti

Sam

plin

g sit

e D

ate

Cya

noba

cter

ia

Wat

er k

gil)

C

ell (

,@I)

Chl

omph

yll a

W

ater

tem

p.

Lake

Sag

ami Ju

ly 2

7. 1

992

Aug

. 12,

1992

A

ug. 2

0,19

92

Sept

3,

1992

Ju

ly 2

9.19

93

July

27.

1994

A

ug 2

3,19

94

Sept

. 16,

199

4 A

ug 2

4,19

95

A

July

27,

1992

A

ug 1

2,19

92

Aug

. 20,

199

2 Se

pt. 3

. 19

92

July

29,

1993

Se

pt. 2

8,19

93

Oct

. 21.

19m

Ju

ly 2

7,19

94

Aug

. 23,

1994

Sept

16,

1994

B

Sept

16,

1994

A

Aug

. 24.

1995

Sept

. 21.

199

5

Lake

Tsu

kui

A.

A.

A. M.a

.M.w

M. a

. A

M. a

. M. w

.

M. a.

M. v

. M

. a.

~ M. a

, M. v

. M

. a, M

. v.

M.a

M.v

M

.a,M

.v

M. a

,M. v

. M w

M

. a

M. a

M

.a.M

.v

M.a

,M. v

.M w

M.a

,M.v

M

.a.M

.v

A..

M a

M

. a. A

.

ND

N

D

ND

N

D

ND

N

D

ND

N

D

ND

0.05

0.

02

0.20

0.

08

0.07

0.

04

ND

0.

15

0.03

ND

2.

64

ND

N

D

ND

N

D

ND

N

D

ND

N

D

ND

N

D

ND

ND

N

D

ND

N

D

ND

N

D

ND

N

D

ND

N

D

00

9

ND

N

D

ND

N

D

ND

N

D

ND

N

D

ND

0.

18

ND

N

D

ND

0.

09

ND

0.

05

ND

0.

34

ND

1.

88

0.04

0.

45

ND

9.

68

0.14

14

3 N

D

13.0

N

D

17.1

N

D

0.04

N

D

0.09

0.

05

8.19

N

D

0.88

ND

0.

10

1.07

37

8

ND

0.

20

ND

0.

03

ND

N

D

ND

ND

N

D

0.07

00

4

0 1

8 0.

62

0.09

N

D

9.70

0.M

2.

65

ND

N

D

0.33

N

D

ND

10

.0

0.03

ND

ND

N

D

ND

N

D

ND

0.

02

0.04

0.

05

0.60

0.08

3.

18

52.0

3.

90

2.50

N

D

ND

1.

78

0.24

0.04

93.8

0.08

0.02

74

47

80

14 5 40

38

38

55

46

441

616

110

762

292 18

676 66

40

6156

23 7

0

28.0

3

22.5

E F1 2 5 %

21

.2

27.5

22

.4

27.5

24

.5

20.5

27

.5

=!

28.0

24

.0

30.1

30

.4

28.6

22

.4

17.0

28

.5

26.0

24.5

24.5

29.8

25.0

* ND

. Mic

rocy

stin

s not

det

ecte

d (4

.02 ,u

g/l);

M. a

.. M

icro

cysh

~ oem

rgim

sa ; M

. v., M

icro

cyst

is vi

ridi

s ; M

. w..

Mic

rocy

stis

wes

enbe

rgii

;

A..

Anab

aena

sp .

(Fr

omTs

uji e

t al.

Natu

ral T

oxin

s. 4.

189

(19%

). W

ith p

erm

issio

n.)

W

W

c.

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6 (2)-Adda Microcystin-LR 6 (2)-Adda Microcystin-RR

R = CH(CH3)z R = CHzCH2NHC(=NH)NHz

Fig. 2 Structures of 6(Z)-Adda microcystins-LR and -RR

water and investigate their stability, lake water (10 1) collected from Lake Tsukui on

September 16, 1994 was permitted to stand at 4 'C in the dark. After 3 days and 7

days, 9% and 15 7% release of microcystins from the cells were observed, respectively.

After 3 days, the lake water was filtered through a glass GF/C filter, and the microcystins

in the filtrate were further left at 4 'C or 20 'C. The concentration was maintained at

almost the same level up to 14 days at 4 'C and only 10% of the microcystins remained

after 42 days (Fig. 2). However, no microcystins were detected at 20 "C after 42 days.

These results are similar to the release and degradation kinetics reported by Jones et al.

(25), Kenefick efal. (26) and Watanabe etal. (27).

As mentioned above, four pathways may be considered to contribute to natural

routes of detoxification of microcystins: adsorption, thermal decomposition aided by

temperature and pH, photolysis and biological degradation. We have examined the

stability of microcystins under photolysis and thermal decomposition conditions (28,29).

Here, the persistence and decomposition of microcystins in the natural environment under

the conditions described above will be discussed based on these results.

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0 10 20 30 40 50

Time (days)

Fig. 3 Release of microcystin from the natural bloom into the water phase and persistence of microcystin in lake water at 4°C in the dark. (FromTsuji et al. Natural Toxins, 4, 189 (1996). With permission.)

THERMAL DECOh4POSITION

The pH and temperature of lake water are usually 9-10 and 2530"C, respectively,

when cyanobacteria occur intensively in the summer season in Japan. Therefore, the

stability of microcystin LR was examined at pH 9 and 21-30°C for 100 days in addition

to other conditions, pH 1,5 and 7, and 5, 20 and 40°C. Fig. 4 demonstrates the effect

of pH and temperature on the stability of microcystin LR. At 5 and 20"C, limited

decomposition was observed at any pH, whereas the half-lives of microcystin-LR were 3

and 10 weeks at pH 1 and 9. respectively at 40°C. The decomposition rate was

slightly decreased under conditions mimicking the natural conditions in summer

compared with that at pH 9 and 40'C. These results indicated that the half-life of

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0 20 40 80 80 Time (days)

0 20 40 60 80 Time (days)

A 5 " C o:20°c

0 20 40 80 80 Time (days)

Time (days)

0: 21 -30 "C 0: 40 "C

Fig. 4 Effect of pH and temperature on the stability of microcystin-LR. (From Harada et al. Phycologiu, 35 (6, supplement), 83 (19%).

With permission.)

microcystin LR was about 10 weeks under conditions mimicking the natural conditions in

summer and the contribution of the thermal decomposition is less than those of other

factors (29).

The structural analysis of degradation products at 40°C was carried out, because

the decomposition proceeded more smoothly than at 21-30°C as shown in Fig. 4. To

elucidate the structures of the degradation products, the reaction mixtures were analyzed

by LC/MS analysis, suggesting that the initial hydrolysis occurs at the Mdha moiety to

give a linearized peptide, because the Mdha has an enamide structure. Successively, it

was decomposed to smaller linearized peptides under conditions used. The degradation

scheme of microcystin-LR is shown in Fig. 5 (29).

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MW

101

2

n W

82

9

R=N

H-C

Hs

" 'OZH

YW

818

R=

OH

,kR

0

H

Fig.

5 D

egra

datio

n sch

eme o

f mic

rocy

stin

-LR

at d

iffer

ent c

ondi

tions

of t

empe

ratu

re an

d pH

(F

rom

Har

ada e

t al.

Phyc

olog

ia, 3

5 (6

. sup

plem

ent),

83 (1

996)

. With

per

miss

ion.

) 0

\o

v,

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-1 00 E I P c e 50

ii E 9

5 O l I I I

0 5 10 15

Time (days)

Fig. 6 Decrease of total microcystin ( November 10-24.1990) by irradiation with sunlight in various pigment solutions. (*) chlorophyll a (20 mgll); (0) B-carotene (20 mg/l);(O) waterextractable pigment (5 mgl1)jA) solvent-extractable pigment (5 mgll); (0) blank. (From Tsuji et al., Envinm. Sci . Techol., 199428,173. With Permission.)

PHOTOLY SIS

Sunlight irradiates the earth at wavelengths above 295 nm and is essential for

growth of cyanobacteria. The idluence of fluorescent light and natural sunlight on

stabilityof microcystin-LR in distilled water was studied for 26 days, but no significant

change was observed. Cyanobacteria possess several pigments for photosynthesis such

as chlorophyll a, p-carotene and phycocyanins. If cells decompose under field

conditions, microcystins would be exposed to sunlight together with coexisting pigments.

Fig. 6 illustrates the decrease in microcystins due to irradiation with sunlight for 15 days

in the presence of various pigments, indicating that the presence of the pigments

accelerates the decomposition. However, no significant decomposition of microcystin-

LRoccurred in pigment solutions on irradiation with fluorescent light (28).

Fig. 7 (a) shows the total ion chromatogram of the photolysis product of

microcystin-LR after 7 days. One new peak (A) appears together with the starting

material and small peaks (X and Y). A co-elution experiment and the mass spectra (Fig.

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HEPATOTOXIC MICROCYSTINS

(a) (b) m a q

R . T . 2p

397

. 5 B(Z) -Adda m l c r o c y s t l n LR p e a k A

( M W 9 9 4 )

+1.0

500 1000 n/z

200

500 1000 n/z

100!l M l c r o c y s l l n ( M W 994) LR

l;;L7

50

[ M I H I + [MIHI+ 10 9 9 5

5 3

500 1000 500 1000 n/z n/z

Fig. 7 Frit-FAB LC/MS analysis of photolysis products of microcystin-LR. (a) Total ion chromatogram and mass chromatograms monitored at mlz 995 and 135: (b) Frit-FAB LC/MS mass spectra of the products. (From Tsuji el al. Environ. Sci. Techno/., 1994, 28, 173. With permission)

7 (b)) indicated clearly that this peak corresponds to a geometrical isomer, the 4(E) , 6(2)

isomer of the diene of the Adda portion of microcystin-LR (Fig. 2). We have isolated

these geometrical isomers of microcystins-LR and-RR in the course of isolating

microcystins from natural blooms of Microcystis (9, 10) Toxic bloom samples usually

contain 5 to 15 8 geometrical isomers. The isolated isomers do not show hepatotoxicity

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0 10 20 Time(days)

Fig. 8 Isomerization of microcystin LR and 6(Z)-Adda microcystin LR (Apnl 12-May 9. 1991). (0) microcystin LR in water-extractable pigment solution; (0) 6(Z)-Adda

microcystin LR in water-extractable pigment solution; (A) microcystin LR in solvent-extractable pigment solution; (A)qZ)-Adda microcystin LR in solvent-extractable pigment solution. Pigment concentration, 5 mg/ml. (From Tsuji et al.. Environ. Sci. Technol., 1994. 28. 173. With Permission.)

(9) and have much weaker tumor promoting activity than their parent toxins (30).

Adda microcystin-LR was also isomerized to microcystin-LR under the same conditions.

6 (a-

The time course of the isomerization for microcystin LR and its isomer is shown in

Fig. 8. Both isomers were gradually isomerized to the corresponding ones and the

reactions reached an equilibrium after 18 days in the presence of water extractable

pigments. The isomerization ratios in equilibrium were approximately 0.55.

Subsequently, the effect of the concentration of water-extractable pigments on the

isomerization was observed for 29 days. Although linear relationships between pigment

concentration and isomerization ratio were obtained after 6 and 8 days , the relationship

was not found beyond 8 days due to complete decomposition of both isomers (Fig. 9).

These results indicated that the decomposition and isomerization of microcystin-LR occur

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.001 I I

0.0 Q.2 0.4 0.6

Z I E+Z

Fig. 9 Isomerization of microcystin-LR (10 mgil) by iradiation with sunlight at various concentrations of water extractable pigment. (From Tsuji el al.. Environ. Sci. Technol.. 1994, 28, 173 With Permission.)

simultaneously under these conditions and that the former is predominant at higher

pigment concentrations. The isornenzation was influenced by the presence of pigment in

water, and its rate was dependent on the concentrahon and the type of pigment (28). As

mentioned above, however, the concentration of chlorophyll a was at most 1 mg/l as

shown in Table 1 and photolysis would contnbute to the decomposition of microcystins

on a limited basia.

ADSORPTION

To our knowledge, there is one paper on the adsorption of microcystin on

sediments. Rapala etal. reported that 13-24 pg of microcystins was adsorbed by 1 ml

of the sediment (31). In our preliminary experiment, we ascertained that microcystins,

particularly more hydrophilic ones such as microcystin-RR, are adsorbed strongly on

sediment. Additionally, i t was found that it is very difficult to recover adsorbed

microcystins from sediments by the usual procedure, suggesting the requirement of an

effective method for this purpose.

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BIOLOGICAL DEGRADATION

Water supplies contaminated with toxic cyanobacteria are often treated with copper-

based algicides. These algicides are effective in lysing cyanobacterial cells but have the

undesirable consequence of releasing intracellular toxins into the surrounding water.

Usually, it is presumed that the toxins are rapidly diluted by uncontaminated water from

the main body of the lake. Jones and On reported an interesting phenomenon in which

microcystins persisted at higher levels (100-1800 pg/l) for 9 days after algicide treatment

of a Microcystis bloom and then almost all toxins rapidly disappeared (32). We and

another group have observed a similar phenomenon in a laboratory experiment (27,33).

Biodegradation of the toxins probably occurs through bacterial action. Very recently,

some bacteria were isolated and degradation products of microcystin-LR using these

bacteria was structurally characterized (34). These results suggest that microbial

degradation contributes greatly to detoxification of microcystins in natural environment.

BIOLOGICAL ACTIVITY OF DEGRADATION PRODUCrS

Throughout these degradation studies, we obtained several degradation products

of microcystins and investigated their biological activities such as hepatotoxicity, protein

phosphatase inhibition activity and mutagenicity. Linearized peptides were mainly

formed through the initial hydrolysis at the Mdha moiety under thermal decomposition

conditions as shown in Fig. 5. Although it was difficult to show their biological

activities due to the limited amounts, they are not expected to have such activities,

because it is found that several linearized peptides of microcystin and nodularin do not

show the original biological activities (11, 34). In the same experiment, the 6-

monomethyl ester (Glu-methyl ester) of microcystin-LR was also obtained, whch did not

show the activities and was consistent with the results of Namikoshi et al (35) and Ann

and Carmichael(36).

Under photolysis conditions, a geometrical isomer of microcystin, 6(Z)-Adda

microcystin, was formed, whch is shown to be non-toxic (9) and not to inhibit protein

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HEPATOTOXIC MICROCYSTINS 40 1

phosphatases 1 and 2A (31). To date, no noxious degradation products have been

detected from microcystins. These results clearly indicate the importance of cyclic

structure, the geometry of Adda and the free carboxylic acid of glutamic acid in the

toxicity of microcystins.

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Rinehart, K. L., Harada, K.-I., Namikoshi, M., Chen, C., Harvis, C. A, , Munroe, M. H. G., Blunt, J. W., Mulligan, P. E., Beasley, V. R., Dahlem, A. M. and Carmichael, W. W., Nodularin, microcystin, and the confifuration of Adda. J . Am. Chem. SOC., 110: 8557, 1988.

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12. Kondo, F., Ikai, Y., Oka, H, Okumura, M., Ishikawa, N.. Harada, K.-I., Matsuura, K., Murata, H. and Suzuki, M., Formation, characterization, and toxicity of the glutathione and cystein conjugates of toxic heptapeptide microcystins. Chem. Res. Toxicol. 5: 591, 1992.

MacKintosh, R. W., Dalby, K. N., Campbell, D. O., Cohen, P. T. W., Cohen, P. and MacKintosh, The cyanobacterial toxin microcystin binds covalently to cystein-273 on protein phosphatase 1, FEBS Letters, 371: 236, 1995.

14 Runnegar, M., Bemdt, N., Kong, S-M., Lee, E. Y. C. and Zhang, L., In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A, Biochem. Biophys. Res. Commun., 216: 162, 1995.

15. Y o 0 RS, Carmichael WW, Hoehn RC, Hrudey SE,:"Cyanobacterial (Blue-Green Algal) Toxins: A Resource Guide." p. 84, Denver: AWWA Research Foundation, 1995.

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17. Falconer, I. R., Smith, J. V., Jackson, A. R. B., Jones, A. a d Runnegar, M. T . C., Oral toxicity of a bloom of the cyanobacterium Microcystis aeruginosa administered to mice over periods up to one year, J. Toxic. Envir. Hlth, 24: 291, 1988.

18. YOSHIZAWA, S., MATSUSHIMA, R., WATANABE, M. F., HARADA, K.-I., ICHIHARA, A., CARMICHAEL, W. W. and Fvm, H., Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J . Cancer Res. Clin. Oncol. 116: 609, 1990.

19. MACKINTOSH, C,. BEATllE, K. A., KLUMNPP, S., C O W , P. and CODD, G. A, , Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 264: 187, 1990.

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21. ERIKSSON, J. E,. TOIVOLA, D., MERILUOTO, J. A. 0.. KARAKI, H. Y-G and HARTSHOME, D., Hepatocyte deformation induced by cyanobacterial toxins reflects inhibition of protein phosphatases. Biochem. Biophys. Res. Commun. 73: 1347, 1990.

22. Nishiwalu-Matsushima, R., Ohta, T., Nishiwaki, S., Suganuma, M., Kohyama, K., Ishikawa, T., Carmichael, W. W . b d Fujilu, H., Liver tdmor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J Cancer Res Clin Oncol 118: 420, 1992.

23. Tsuji, K., Naito, S., Kondo, F., Watanabe, M. F., Suzuki, S., Nakazawa, H., Suzuki, M., Shimada, T., Harada. K.-I., A clean-up method for analysis of trace amounts of microcystins in lake water. Toxicon 32: 1251, 1994.

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28. Tsuji, K., Naito, S . , Kondo, F., Ishikawa, N., Watanabe, M. F., Suzuki, M. and Harada, K.-I., Stability of microcystins from cyanobacteria: Effect of light on decomposition and isomerization. Environmental Sciences Technology 28: 173, 1994.

29. Harada, K.-I., Tsuji, K., Watanabe, M. F. and Kondo, F., Stability of microcystins vrom cyanobacteria-I11 Effect of pH and temperature, Phycologia 35: 6 supplement, 83, 19%.

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31. Rapala, J., Lahti, K., Sivonen, K.and Niemela, S. I. , Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a, Letter in Applined Microbiology 19: 423, 1994.

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Documents

Persistence and Decomposition of Hepatotoxic Microcystins Produced by Cyanobacteria in Natural Environment