Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp.: structure of a new hepatotoxin

0041-0101/94 56.00+ .00 © 1993 n Reu Ltd

Toxknn, Vd . 32, No. I, pp . 133-139, 1944. Riated in Graat Hritain.

ISOLATION AND IDENTIFICATION OF 12 MICROCYSTINS FROM FOUR STRAINS AND TWO BLOOM SAMPLES OF MICROCYSTIS SPP.: STRUCTURE OF A NEW HEPATOTOXIN RAUA LwxxAIrii~r,` MICHIO NAMIxosI~n, 2 KAwxINA SnroNSN, `" ICsNN>;rx L. RnvExAIS~ and SEPPO I. Nii~tiELÀ`

'Department of Applied Chemistry and Microbiology, P.O. Box 27, FIN-00014 University of Helsinki, Finland; and'Department of Chemistry, University of Illinois, Urbane, IL 6(801, U.S .A . (Received

6 July 1993 ;

accepted

6 September 1993)

R. LUUICKAINEN, M. NAl~cosrn, K. SIVONEN, K. L. RINEHART and S. I. Nn~Ln . Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp .: structure of a new hepatotoxin . Toxicon 32, 133-139, 1994.~ixteen microcystins, cyclic heptapeptide hepatotoxins, were isolated and purified by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from four hepatotoxic strains and two Microcystis spp. bloom samples originating from five different lakes in Finland . The structures of a new [Dha~MCYST-FR and 11 known microcystins MCYST-LR, [D-Asp~]MCYST-LR, [Dha~MCYST-LR, [n-Asp 3,Dha~ MCYST-LR, MCYST-RR, [D-Asp 3]MCYST-RR, [DhaTJMCYST-RR, [nAsp3,Dha~MCYST-RR, [L-Ser~JMCYST-RR, MCYST-YR and [Dha~ MCYST-YR were assigned based on amino acid analysis, fast atom bombardment mass spectrometry (FABMS) and tandem FABMS. Four other new compounds allowed only determination of their molecular formulas and amino acid components because of inadequate amounts obtained . [Dha~MCYST-RR was found most frequently in these samples as the main toxin .

MASS occurrences of cyanobacteria (blue-green algae), water blooms, are worldwide phenomena in eutrophicated fresh and brackish water bodies. The toxicity of water blooms has been known for over one-hundred years (FxANCts, 1878) . Toxic cyanobacteria cause fatal poisonings of mammals, birds and fish (CARMICIiAEL, 1989, 1992; Conn and Pooh, 1988; $KULBERG et al., 1984) and may also cause adverse effects on human health (BOURKE and HAwFS, 1983; FALCONER et al., 1983; GORHAM and CARMICHAEL, 1988; $CHVVIMbIEIt and Scxwn~, 1968). Cyanobacterial toxins are classified into two main groups : hepatotoxic peptides (microcystins and nodularin) and neurotoxic alkaloid-like wmpounds (CARMICHAEL, 1989, 1992; Conn and Pooh, 1988) . Recently, microcystins and nodularin have been shown to be protein phosphatase inhibitors (PPl and PP2A) (MACKINTOSH et al., 1990; MATSUSI~uMA et al., 1990) and potential tumour promoters (NISHIWAKI-MATSUSfmKA et al., 1992) . Author to whom correspondence should be addressed. 133

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Microcystins are monocyclic heptapeptides composed of >}alanine (p-Ala)', two variable L-amino acids at positions 2 and 4, ß-linked D-erythro-ß-methylaspartic acid (D-McAsp) 3, the novel ß-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl10-phenyldeca-4,6-dienoic acid (Adda)S, y-linked n-glutamic acid (D-Glu°, and Nmethyldehydroalanine (Mdha)' (CARMICHAEL et al., 1988). Over 40 different varieties of microcystins have been identified to date (LUUKKAINEN et al., 1993 ; Nwn~uICOSFU et al., 1992b, c, d; SIVONEN et al., 1992x, b, c, d). The known freshwater cyanobacterial genera producing microcystins are Microcystis (e .g. BOTES et al ., 1984, 1985 ; KusuMl et al., 1987 ; NAM>zcost-n et al., 1992x ; ScoTT, 1991 ; SI-ultwl et al., 1991 ; Slvoxsx et al., 1992c; WwTwxwsE et al ., 1988), Anabaena (Hwitwnw et al., 1991x ; KRISHNAMURTHY et al., 1986; SIVONEN et al., 1992x), Oscillatoria (KRISI-nvwMuRTHx et al., 1989 ; LwlcxwnvEx et al., 1993 ; MERILUOTO et al., 1989) and Nostoc (NwMncosFn et al., 1990 ; SIVONEN et al., 1992b) . In brackish water habitats Nodularia spumigena has been found to produce nodularin, a cyclic hepatotoxic pentapeptide closely related to microcystins (RIIJEHART et al., 1988 ; $IVONEN et al., 1989). In this study, we report the isolation of 16 microcystins from two bloom samples and four strains of Microcystis spp. from five different Finnish lakes. The structures of one new toxin and 11 known microcystins were assigned based on the amino acid analysis and fast atom bombardment mass spectrometry (FABMS), including tandem FABMS (FABMS/CID/MS) (NAMIK0 .4HI et al., 1990, 1992x). The bloom samples were collected and strains isolated from lakes in Finland during summers in 1986 and 1987 (Table 1) . Microcystis aeruginosa strain 205 was isolated from the corresponding M. aeruginosa bloom. The other bloom sample studied (116) consisted of M. wesenbergü and M. aeruginosa. The strains were grown in Z8 medium in batch cultures at room temperature under continuous illumination of about 50 micrceinsteins/mZ/sec as detailed earlier (Sivoxsly et al., 1990). Cells were harvested after 14-16 days of incubation and lyophilized prior to toxicity testing and toxin isolation. Toxins were Tea~ 1 . FABMS DATA, TFIE RELATIVE s~rLe exn sTttucruxe wss
Toxin A B C D

M+H

Straia

RAI70 (%) OF L40LATFD TOXIN COMPONENIS POR EACH m~aNS A-P >sou~o t~ Aflcrocystic srxeuvs ~wn

ai oos~s

Bloom

Assignment 967 6 [o-Asp3,Dha~]MCYST-LR 981 69 2.5 16 [Dha~MCYST-LR 981 1 [o-Asp~]MCYST-LR 995 21 1 MCYST-LR E 1010 11 [o-Asp',Dha~]MCYST-RR F 1015 1 [Dha~MCYST-FR (NEV1~ G 1024 8 95 66 88 96 [Dha~]MCYST-RR H 1024 3 3 [o-Asp~]MCYST-RR I 1031 9 [Dha~MCYST-YR J 1035 1 unknown K 1038 96 MCYST-RR L 1040 1 l unknown M 1042 l 1 1 1 [t-Ser~MCYST-RR N 1045 I MCYST-YR O 1049 1 unknown P 1058 2 .5 I 1 1 unknown Strain or bloom originates from ' L. Pyhäjärvi, f L. Hirsijärvi, $ L. Rusutjärvi, § L. Mallusjürvi, and ~~ L. Pitkäjärvi, Finland. m/z

98'

136t

199$

205§

116~~

205§

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13 5 7 Mdha (Dha) (L-Sar)

6 D-Glu

R~

RZ

R3

X

A

H

H

CII Z

L-Leu

B

CH 3

H

CH Z

L-Leu

C

H

CH 3

CH Z

L-Leu

D

CH 3

CH 3

H

H CH 3

H

CH 3

H

CH Z

L-Lcu

CII Z

L-Arg

CH Z

L-Phe

H

CIi Z

L-Arg

H

CH 3

CII Z

L-Arg

I

CH 3

H

CH Z

L-Tyr

K

CH 3

CH 3

CIi Z

L-Arg

M

CH 3

H

H, CH ZOII

L-Arg

N

CH 3

CH 3

CH Z

L-Tyr

F G

H

FYa . 1 . T~ srxuctvn~ oF ~ncxoc~rsnNS (A-I, K, M erm N) tso~zm Fxox MicrocystLs spp. sr~uns nrm er oo~s .

extracted with 5% acetic acid (HARwnA et al., 1988) and isolated by repeated ODS HPLC and silica gel TLC as detailed earlier (LUUKKAINEN et al., 1993) . Sixteen microcystins were isolated, as listed in Table 1 . All Microcystis samples contained several hepatotoxins (Table 1). The most common toxin, found in five samples and dominant in four cases, was [Dha~]MCYST-RR (toxin G), the amounts of which varied between 5 and 3545 hg/g of freeze dried cells. Microcystis strain 98 produced [Dha~MCYST-LR (toxin B) as the main toxin, and bloom sample 116, consisting of M. wesenbergü and M. aeruginosa, was shown to contain mostly MCYST-RR (toxin K). Toxins A-E, G, H, K and N were assigned the structures shown in Fig . 1 by FABMS and TLC compared with authentic samples isolated previously from Microcystis spp. (Nwl~cosH1 et al., 1992a) and Ana6aena spp . (SIVOIVEN et al., 1992a) . Toxin I showed a molecular ion peak at m/z 1031.5231 (M + H, CS,H N,°0,3, 0 -2.9) in the HRFABMS obtained with a matrix of dithiothreitol/dithioerythritol

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(`magic bullet' ; Wrrrex et al., 1984). Toxin I showed D-MeAsp together with n-Ala, r.-Tyr, t,-Arg, and n-Glu analyzed by chiral capillary GC which revealed the structure of toxin I as [DhaTJMCYST-YR (Fig. 1 ; Snroxsx er al., 1992c). Similarly, toxin M (m/z 1042.5693, M + H, C,sHN 0,3 0 -0.7, HRFABMS ; n-Ala, L-Arg, o-MeAsp, ~-Arg, n-Glu, z-Ser) was assigned as [L-Ser~]MCYST-RR as shown in Fig. 1 (NAMIIC06HI et al., 1992c). Toxin O was the same as the microcystin isolated from a water bloom of Microcystis spp . collected from Homer Lake (Illinois) (N~cosi-n et al., 1992a) . The four toxins F, J, L, and P were new compounds since their mol . wts are not identical to known microcystins . Although the amounts of these toxins isolated were very small, toxin F has been characterized from its mass spectral and amino acid analysis data. Toxins J, L, and P allowed only determination of the molecular formulas and amino acid components: toxin J, n-Ala, n-McAsp, t-Arg, and n-Glu, m/z 1035.5536 (M + H), CS,H,SN,o0,3, O -2.1; toxin L, n-Ala, L-Arg, D-McAsp, z-Arg, and n-Glu m/z 1040.5540 (M + H), C,sHNO, 0 -1 .1 ; toxin P, o-Ala, z-Arg, n-McAsp, z-Arg, and n-Glu m/z 1058.5650 (M + H), C~H,6N0,4, O -1 .5. Toxin F from M. aeruginosa strain 98 showed a molecular ion peak at mlz 1015.5283 (CS,H,~N,oO,Z, O -3.0) by HRFABMS . The amino acid analysis on chiral capillary GC revealed n-Ala, z-Phe, n-McAsp, ~-Arg, and n-Glu, suggesting that toxin F is a demethyl variant of MCYST-FR (mol. wt 1028) (N~a~tncosi-n et al., 1992a) . FABMS/CID/MS (Nnauxosi-n et al., 1990, 1992a) showed fragment ion peaks at m/z 135 [PhCHZCH(OCH 3)] and 879 (M - 135), which proved the presence of the Adda unit (NAMIROSHI et al., 1992a) . Fragment ion peaks at m/z 361 (C H, SO-GIuDha), 199 (Glu-Dha + H), and 141 (Dha-Ala + H) were observed, each 14 Da less then the corresponding peaks of MCYST-FR (at m/z 375, 213, and 155, respectively) (NnamcosFn et al., 1992a) and the sequence of Adda-Glu-Dha-Ala was identified from these fragment ions. The fragment ion peak of (MeAsp-Arg + H) was detected at m/z 286. Consequently, the structure of toxin F was assigned as [Dha~JMCYST-FR, as shown in Fig . 1. Toxin F was hepatotoxic to mice when tested in pure form but the amount was too small for quantitative determination of its toxicity. Microcystis aeruginosa is the most common producer of microcystins : toxins of bloom samples (CARMICHAEL et al., 1988; EL~MnN et al., 1978; Hnxnnn et al., 1990; Kxisxxn~ntz'xx et al., 1989; NAMIICOSHI et al., 1992a, d; Sco~-r, 1991 ; Sfmtni et al., 1991 ; WATANAHE et al., 1989; Zxwxo ei al., 1991) and of Microcystis strains (Booms et al., 1984, 1985; H~tnnn et al., 19916 ; KivmnxTn et al., 1992; KRisxxa~uxTxx et al., 1986; Kuxcsuwnx et al., 1988; $HIRAI et al., 1991 ; $IVONEN et al., 1992c; WATANABE et al., 1988, 1991) have been characterized . MCYST-LR is found to occur most frequently among the microcystins in the samples originating from different parts of the world (Bo~rES et al., 1985; CARMICHAII, et al., 1988; KRLSHNAMURTHY et al., 1989; Kuxcsuwnx et al., 1988; N~a~ucost-u et al., 1992a; WATnxnaE et al., 1988, 1989, 1991) . In this study it was found in one bloom (116) and one strain sample (98) but as a minor component . In addition to MCYST-LR, MCYST-RR and MCYST-YR have been found frequently in M. aeruginosa samples obtained from South Africa (Bo'rt=s et al., 1985), Japan (H,utnnn et al., 1990; Kuxcsuwnx et al., 1988; WATANAHE et al., 1988, 1989, 1991), China (CARMICHAEL et al., 1988; ZHANG et al., 1991), and the U.S.A. (Nnruicosr-n et al., 1992a) . Most of the M. aeruginosa and M. viridis strains and several bloom samples systematically studied in Japan contained mixtures of MCYST-LR, MCYST-RR and MCYST-YR (Si-mini et al., 1991 ; W~T~x~sE et al., 1988, 1989, 1991). In the present study, only one bloom sample (116) was found to contain MCYST-RR as the main toxin . In other samples the major component was most frequently [Dha~JMCYST-RR and occasionally [Dha~]MCYST-LR .

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Other demethyl variants of MCYST-LR, MCYST-RR or MCYST-YR were found as minor components. Finnish Oscillatoria agardhü strains produced only demethyl microcystins (LUUKKAINEN et al., 1993), which were also common among Anabaena spp. strains (Snroxaly et al., 1992x) originating from the same geographical area. Recently, demethyl microcystins ([Dha~JMCYST-LR and [n-Asp',Dha~MCYST-LR) have also been reported in some strains of M. aeruginosa collected from Japan (HARADA et al., 1991b; $HIRAI et al., 1991) and [nAsp']MCYST-LR from a bloom sample (HARADA et al., 1990), but demethyl microcystins seems to be more common among cyanobacteria in Scandinavia. One M. aeruginosa strain isolated from Russian Karelia produced only demethyl microcystins (SIVOxEx et al., 1992c) . Interestingly, MCYST-YR or its demethyl variants have been found so far only in Microcystis spp. (BOTES et al., 1985 ; NAi~coslu et al., 1992x; ScoTT, 1991 ; $HIItAI et al., 1991 ; $IVONEN et al., 1992c; WATANABB et al., 1988), not in any other cyanobacterial genera . In the present study several new minor compounds were found in the genus Microcystis. The structures remain unsolved because ofinsufficient amounts of toxin available. The new hepatotoxin, [Dha~JMCYST-FR, found in this study is the first reported demethyl variant of MCYST-FR. Phenylalanine as an amino acid component of microcystins has been found so far in several samples from South Africa (Score, 1991), a Homer Lake sample from the U.S.A . (NAMIIC06FII et al., 1992x) and a bloom sample from Finland (this study) . Of the known toxins, this is the first time that [L-Ser~JMCYST-RR has been found in a Microcystis sp. It has been reported previously only in a Finnish Anabaena strain (NAruxosfn et al., 1992c) . Studies ofbiogenesis and genetics of cyanobacterial microcystins will be needed to reveal why so many varieties of these compounds are produced at a time and why certain compounds are found as the major toxins . Acknowkdgementa-Research at the University of Helsinki was supported by grants from the Academy of Finland, The Maj and Tor Neasling Foundation, and the University of Helsinki, and at the University of Illinois by grants from the National Institutes of Allergy and Infectious Diseases (AI 04769) and of General Medical Sciences (GM 27029), both to K.L.R . We thank Mr F. SuN for mess spectra and Ms Renee $AASrAMOINEN for technical assistance in culturing the cyanobacteria. REFERENCES

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(1992b ) Structures of three new homotyrosine-containing microcystins and a new homophenylalanine variant from Anabaena sp. strain 66. Cltem . Res. Toxicol. S, 661-666. Nwt~mcosxi, M ., $rvONEN, K., Evwxs, W. R ., CARMICHAQ., W . W ., S~x, F., Routnwtxerr, L ., Luuxtwxt:x, R. and Rtrt~twRr, K . L. (1992c) Two new t.-serine variants of microcystins-LR and -RR from Anabaerta sp . strain 202 A1 and 202 A2 . Toxicon 30, 1457-1464 . Nwt~eosin, M ., Srvoxt=.x, K ., Evwxs, W. R ., Sux, F., Cwte~acxwr;r.., W. W. and Rnvniwxr, K . L. (19924) Isolation and structures of microcystins from a cyanobactetial water bloom (Finland). Toxicon 30, 1473-1479 . Ntst~mvwtct-Mw~tsustßsiw, R ., Otrrw, T., Ntstnwwtct, S ., Suawxu~w, M ., Konxwesw, K., Isrmcwww, T., Cwtuttcttwta., W. W. and Fume, H . (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res . Clin . Oruol. 118, 420-424 .

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Rnuttexr, K . L ., Heaene, K: L, Ne~os~, M ., Ct~tv, C., Hwsvts, C. A., Muxtro, M . H . G ., BLUNT, J . W ., MuLLtceN, P . E ., BEASLEY , V . R, DAtuFat, A. M . and CARMICHAII. W . W. (1988) Nodularin, microcystin, and the configuration of Adda. J. Am . them . Sce. 110, 8557-8558 . $CNWrèO~it, M . and Scxwmta~te, D . (1968) Medical aspects of phycology. In: Algae, Man, and the Ermironment, pp. 278-358 (IACIC~ON, D. J ., Ed.) . Syracuse NY: University Press . Scow, W. E . (1991) Occurrence and significance of toxic cyanobacteria in Southenn Africa. Wat. Sci. Tech. 23, 175-180. Starter, M ., Oxret~, A ., SeNO, T., Me~stnaoro, S ., Setce~oro, T., Sero, A ., Acne, A ., Hettene, K :L, Staitene, T ., Suzutu, M . and NA¢etvo, M . (1991) Toxicity and toxins of natural blooms and isolated strains of Microcystls spp . (cyanobacteria) and improved procedure for purificatioa of cultures . Appl. Enoiron. Microbiol. 37, 1241-1245 . Srvotveiv, K., Koxorierr, K ., Cewactteta  W . W., DAttLl :~r, A. M ., Rt r, K. L., ICtvaterrre, J. and N»~m.'C, S . L . (1989) Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and the structure of the toxin . Appl. Ewbon . Mkrobtol. SS, 1990-1995 . Srvo~nr, K., N~.,x, S. L, Ntt~n, R . M ., LT:rtsrb, L ., Luosre, T . H. and R~r~v, L . A . (1990) Toxic cyanobacteria (bluo-green algae) in Finnish fresh and coastal waters. Hydrohiologia 190, 267-275. Srvormv, K., Ne~osHI, M ., Evens, W. R ., CARMICHAEL, W. W., SuN, F ., ROUFUetNIIV, L ., Lwittcetxeiv, R . and RtN~texr, K. L. (1992x) Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena. Appl. Eroiron. Microbiol. 58, 2495-2500. Srvor>En, K ., NA~ostn, M ., Evens, W. R ., FAantc, M ., Ceteeacxe>?t ., W. W . and RrrrEttear, K. L. (1992b) Three ntw microcystins, cyclic heptapeptide hepatotoxins, from Nostoc sp. strain 152 . Chem . Res. Toxkol. S, 464-469 . Srvoxw, K ., NeatAOSta, M ., Evens, W . R ., Gxotarn, B. V., CAMaCHAEL, W. W. and RIrirtteRr, K. L . (1992c) Isolation and structures of five microcystins from a Russian Mkrocystis aeraginosa strain CALU 972 . Toxicon 30, 1481-1485 . Srvormv, K., Strut.aFne, O . M., NeiaßosFn, M ., Evens, W. R., Cea~cttetz W . W. and Rtxatterer, K . L . (19924) Two methyl ester derivatives of microcystins, cyclic heptapeptide hepatotoxins, isolated from Anabaena floc-aquae CYA 83/1 . Toxkon 30, 1465-1471 . Sxur.at:ac, O . M ., Coon, G . A. and Cett~cxe8,, W . W. (1984) Toxic bluo-green algal blooms in Europe : a growing problem. Ambio 13, 244-247. WATANAItE, M . F., Otstfl, S., Hesene, K.-L, Mersuuxe, K., Kwwet, H . and Suztrat, M . (1988) Toxins wntained in Microcystis species of cyanobacteria (bluo-green algae) . Toxicon 26, 1017-1025 . Wereiveat:, M . F., Hetune, K :L, Merswtu, K ., Outs, S., W~rexAnF Y. and Suzvtu, M . (1989) Heptapeptide toxins contained in natural samples of Microcystis species . Toxklty Assessment 4, 48797 . WATANAtlE, M . F., WATANAttPy M ., Iüro, T., Hettene, K :I. and Suzua, M . (1991) Composition of cyclic toxins among strains of Mkrocystis aeruginosa (bluo-green algae, cyanobacteria). Bot. Mag. Tokyo 104, 49-57 . Wrr~v, J . L., Scxet+t~ea, M. H ., O'SttPw, M ., Coots, J . C., Htaa.txc, M . E . and Rtr>Etteter, K. L ., Jtc (1984) Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry . Biochem . Btophys. Res. Commw . 124, 350-358. Z.ttervc, Q:X., Cete~c~teaL, W. W., Yu, M : J. and La, S.-H. (1991) Cyclic peptide hepatotoxins from freshwater cyanobacterial (bluo-gr~oea algae) waterblooms collected in Central China . Frwiron. Toxicol. Chem. 10, 313-321 .