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Microcystin-like toxins in different freshwater species of Oscillatoria
Taxlcon, Vd. 30, No . 10, pp. 1307-1311, 1992. Ainled in Oreal arilain .
0041-0101/92 SS .00 + .00 ® 1992 Pagrmon Aes Ltd
MICROCYSTIN-LIKE TOXINS IN DIFFERENT FRESHWATER SPECIES OF OSCILLATORIA Mu,ENA BRUN0, 3 PAOLA MARGiIBRITA BIANCA GUCCI, 3 ELIO PIERDOMINICI,2 PAOLA SPSTILI, 2 ALFREDO IOPPOL0, 2 NICOLA SECHI3 and LAURA VOLTERRA 3 ~ Departments of 'Environmental Hygiene, and =Veterinary Medicine, National Health Institute, v. le Regina Elena 299, 00161 Rome, Italy; and'Department of Botany, University of Sassari, v. Muroni 25, 07100 Sassari, Italy (Received 9 December 1991 ; accepted 31 Marck 1992)
M. BRUNO, P. M. B, Guccl, E. PIERDOMINICI, P. SESrti.I, A. IOPPOLO, N. SECt~n and L. VoLrERRA . Microcystin-like toxins in different freshwater species of Oscillatoria. Toxicon 30, 1307-1311, 1992.-In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tennis and Oscillatorla mougetü were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms . Tolac blooms are produced by the marine and freshwater genus Oscillatoria (phylum Cyanophyceae) (MOORE, 1982; $KULBERG et al., 1984) . Hepatotoxic eßects (ERIKSSON et al., 1988a; BERG et al., 1988) were described in the case of Oscillatoria aghardü blooms found in lakes in Norway and Finland (Os~rwsvuc et al., 1982; BERG et al., 1986; ERIR3SON et al., 1986). The chemical characterization of hepatotoxins isolated from the CYA NIVA 38 strain of O. aghardü (Norway) (SKULBERG and SKULBERG, 1985) showed marked similarity to the microcystin RR (arginine-arginine variant) (W. CARMICHAEL, Wright State University, Dayton, OH, personal communication) previously described for the toxic components of Microcystis aeruginosa (ERIK3SON et al., 19886). At present, O. aghardü is the only species among freshwater Oscillatoriaceae to have been chemically investigated for toxic blooms (SKULBERG et al., 1984; MERILUOTO et al., 1989). In Italy, toxic blooms of Oscillatoria rubescens were first reported in 1985, in a reservoir system used in Sardinia for the production of drinking water (VOLTERRA et al., 1985; Lolzzo et al., 1988). Following this report, we detected other toxic blooms of the genus Oscillatoria throughout the country (unpublished observations) . The present work is based on the chemical identification of microcystin-like toxins in toxic blooms of various Oscillatoria species, other than O. aghardü, which have been found in the Italian regions of Sardinia and Lazio . Author to whom correspondence should be addressed . 1307
Short Communications
1308
TeeLE 1. Toxcc veLVE (EC,~ oN MICROTOx) eND Toxuv ooNrrffvr (oN HPLC) oF THE THREE aLOOt~rs FROe~ rHE ITALIAN LAEF3 AND OF THE 9TRAIN OF OOMPARLION
Sample/species
Lake/year
TOxin COAient (m8/g dry cells)
ECm
(mg dry cells/ml) -LR like
(1) O . aghardü CYA hTIVA 38 (2) O . teraris +O . r'ubescens (3) O. terrwrs +O. nwugetit
+O.
rubescercs
(4) O . rubeacens
-RR like
-YR like
(Gjersjon, 198~
0 .28
-
2 .90
-
(Simbirizzi, 1989)
2.12
-
0 .48
-
(Flumendosa, 1989) (San Puoto, 1990)
1 .91 0.40
-
-
0.22
1 .16
See text .
The following blooms were examined : (1) mixed blooms of Oscillatoria tennis and O. rubescens (6 .5 x 10° and 7 x 10~ trichomes/litre, respectively, Lake Simbirizzi, January 1989); (2) mixed bloom of O. tennis, O. mougetü and O. rubescens (with a dominance of the species Oscillatoria tennis, Lake Flumendosa, September 1989); (3) O. rubescens bloom (Lake San Puoto, Lazio region), where this species represented 99% of the entire biomass, March 1990. Toxicity tests were performed using lyophilized algae. After extraction of 10 to 200 mg of material (MERILUl7T0 and ERIKS80N, 1988), the samples were tested with a Microtox .d., Microtox Application Notes: system (Microtox Operation Book, Microbics Inc. F M 103-116; U.S. Environmental Protection Agency, 1979). The results were processed statistically on a computer to give the sc~ 5 min value (extinction coefficient of 50% of light emission, after 5 min of exposure to the toxic substance) . The aplysiatoxins test (extraction according to MYND1~tSSE et al., 1977) was performed on female nude mice HRS/J and New Zealand rabbits, as per WHO (Organisation Mondial de la Santè, 1986); experiments were carried out according to the institutional laws on humane care of the animals . Drops of various quantities of the extract (10 ~l for mice and 10 ml for rabbits) were placed on shaven portions of the dorsal skin, and subsequently the animals were checked for cutaneous rashes for an entire week . The chemical identification of toxins was carried out by an HPLC Beckman System Gold instrument, using a Regis column ISRP GFF SS-80 (MERII,UOTO and ERIxssolv, 1988) and a C18 RP Lichrochart column (ERIKSSON et al., 19886), using a mobile phase of 12% acetonitrile/buffer 0.1 M potassium dihydrogen phosphate, pH 6 .8, flow rate 1 mlJmin, wavelength 238 nm with 0.005 AUFS and sample volume loaded 50 pl . Results shown here refer to the Regis column, which separates the RR, YR and LR microcystins used as standards (M131tII,UOTO et al., 1990). The microcystin standards were very kindly provided by Dr M. WATANABB (Metropolitan Research Lab. of Public Health, Tokyo, Japan - -YR tyrosine-arginine variant and -RR arginine~arginine variant microcystins) and Dr W. CARMICHAEL (Wright State University, Dayton, OH, U.S.A . - -LR leucine-arginine variant microcystin) . The samples of the lyophilized strain of O. aghardü ~CYA NIVA 38 (derived from Lake Gjersjon, Norway), were generously donated by Dr J. Eltlxssox (Ä6o Academi, Finland), and were primarily used as comparison for qualitative and quantitative measurements of the toxic peak of the HPLC .
Short Communications
m cuc a ô aN Q
1309
u c
0 a ô
0
S
Time
T
10
15
(min)
(E)
0
S
,
Tlme
10
(mln)
-YR
-LR 0 u C O
a O
aN a
-RR
5
Tlme
(mln )
FiCi . 1 . Ct~tOMA1'OßRAPHIC ANALY~9. OsciUatoria gghardü (red var .) CYA NIVA 38 strain (Lake Gjersjon, Norway) ; (H) O . rubescerts, O. teraris (Lake Simbirim, Sardinia ngion), responsive for -RR atendard; (C) O . rubescens (red var.) (Lake San Puoto, Lazio region); (D) O . rubescens, O . tertuis, O. »tmgetü (Lake Medio F7umendosa, Sardinia region), responsive for -YR standard, compared with the profile of standard toxins . (H) Comparative HPLC profile for standard toxins. Arrows iadicate the toxic peaks . (A)
15
1310
Short Communications
All of the Oscillatoria rubescens algal blooms, tested by the Microtox system, showed values ranging from 0.40 mg/ml (O . rubescens from Lake S. Puoto) to 2.12 mg/ml (O. rubescens from Lake Simbirizzi) (Table 1). The HPLC analyses showed an RR-like toxin for O. tennis plus O. rubescens blooms from Lake Simbirizzi, and a YR-like toxin for O. rubescens from Lazio region as well as from the mined population of O. temtis, O. mougetü and O. rubescens from Sardinia (Table 1, Fig. 1). A previous extract of Oscillatoria rubescens collected from Lakes Mulargia and Flumendosa, in Sardinia, during 1985, and stored at -80°C, gave the same RR-like peak as the bloom tested from Lake Simbirizzi (data not shown). Because of the repeated appearance of non-toxic blooms of Oscillatoria tennis in Lake Simbirizzi (four cases were examined from 1989 to 1990) and because of the presence of an RR-like toxin in a quasi pure bloom of O. rubescens (1985) from Lake Mulargia, the presence of a YR-like toxin in another bloom from Lake Mulargia in September 1989 is thought to be due to the O. mougetü component. Tests performed to verify whether the observed blooms of Oscillatoria could be responsible for cutaneous rashes similar to those caused by aplysiatoxin were negative (MOORS, 1982). Previous chemical studies of toxic Oscillatoriaceae have used the species O. aghardü. In its chemical characterization, the main toxic principle was microcystin-RR (ERIKSSON et al., 19ß8b; MERILUOTO et al., 1989), and therefore this kind of toxin is expected also in other species of Oscillatoria . However, the extract of the bloom of O. rubescens (red var.) from Lake S. Puoto (Lazio region) in March 1990 revealed the presence of a YR-like toxin, and the same was reported for a bloom of Oscillatoria dominated by the species O. tennis, O. mougetü and O. rubescens from Lake Fhimendosa (Sardinia) during September l9ß9 . The presence of hepatotoxic microcystins in various freshwater species of the genus Oscillatoria is not just an ecological problem (ERIICSSON et al., 1986; LINDHOLM et al., 1988). When waters are used for drinking purposes (as in the case of Simbirizzi-Fhurlendosa) it becomes a human health problem. During the potabilization process, Oscillatoria trichomes may be disrupted, and their hepatotoxins released into the drinking water (VOLTERRA et al., 1990). Notwithstanding the taxonomic differences existing between Mycrocystis and Oscillatoria, the production of similar toxins in the two genera raises interesting questions about the evolution of genetic systems showing the same biosynthetic pathway. Ec~
REFERENCES
Besa, K., Setn.eaea, O. M., Sxur aHta, R., UxnESnN., B. and Wn.irrv, T. (1986) Observations of toxic bluo-green algae (Cyanobactetia) in some Scandinavian lakes. Acta Vet. Stand. 27, 440-452. Bma, K., Wnutv, J., C~cxwer., W. and Deaxot .tun, A. (1988) Isolated rat liver perfusion studies with cyclic heptapeptide toxins of Microcystis and Oscillatoria (freshwater cyanobacteria) . Toxicon 26, 827-837. Esncssox, J. E., Mtoeu.uoro, J. A. O. and LQroxot.st, T. (1986) Can cyanobaoterial peptide toxins accumulate in aquatic food chains? Pros. 4th Int. Symp. Microbial Ecol. Ljubljana (Yugoslavia), pp. 655-658 (Meaus~a, F. and Guv'r~tt, M., Eds). Ljubljana: Slovene Society for Microbiology Press. ErutcssoN, J. E., Mettu.uaro, J. A. O., Kurnw, H. P., Jeep. Ar.-LeYr.K, K. and CODD, G. A. (19ß8a) Cellular effects of cyanobacterial peptide toxins . Toxicity Assess. 3, 511-517 . Esurssox . J. E., Mmur.utmo, J. A. O., KUS~w, H. P. and Stctn.r~c, O. M. (198ßb) A comparison of toxins isolated from the cyanobacteria OsciUatoria aghardii and Mlcrceystis aerugtnosa. Comp . Biothem. Physiol. ByIC, 207-210. Lu~mt~tor.x, T., Exnss9ox, J. E. and MESn utrro, J. A. O. (1988) Toxic cyanobacteria and water quality problems . Examples from a eutrophic lake on Aland, SW Finland. Woer Res. 23, 481~ß6. Lotzzo, A., SscFn, N., Vor r~ew+, L. and Cormr, A. (1988) Some features of a bloom of Oscillatoria rulxscens D.C. registered in two Italian reservoirs . Water, Air, SoA Poll. 38, 263-271.
Short Communications
131 1
Mtatn,uoro, J. A. O. and Eturssox, J. E. (1988) Rapid analysis of cyaaobacterial peptide toxine . J. Chromas . 4~, 93-99. Mta
0041-0101/92 SS .00 + .00 ® 1992 Pagrmon Aes Ltd
MICROCYSTIN-LIKE TOXINS IN DIFFERENT FRESHWATER SPECIES OF OSCILLATORIA Mu,ENA BRUN0, 3 PAOLA MARGiIBRITA BIANCA GUCCI, 3 ELIO PIERDOMINICI,2 PAOLA SPSTILI, 2 ALFREDO IOPPOL0, 2 NICOLA SECHI3 and LAURA VOLTERRA 3 ~ Departments of 'Environmental Hygiene, and =Veterinary Medicine, National Health Institute, v. le Regina Elena 299, 00161 Rome, Italy; and'Department of Botany, University of Sassari, v. Muroni 25, 07100 Sassari, Italy (Received 9 December 1991 ; accepted 31 Marck 1992)
M. BRUNO, P. M. B, Guccl, E. PIERDOMINICI, P. SESrti.I, A. IOPPOLO, N. SECt~n and L. VoLrERRA . Microcystin-like toxins in different freshwater species of Oscillatoria. Toxicon 30, 1307-1311, 1992.-In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tennis and Oscillatorla mougetü were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms . Tolac blooms are produced by the marine and freshwater genus Oscillatoria (phylum Cyanophyceae) (MOORE, 1982; $KULBERG et al., 1984) . Hepatotoxic eßects (ERIKSSON et al., 1988a; BERG et al., 1988) were described in the case of Oscillatoria aghardü blooms found in lakes in Norway and Finland (Os~rwsvuc et al., 1982; BERG et al., 1986; ERIR3SON et al., 1986). The chemical characterization of hepatotoxins isolated from the CYA NIVA 38 strain of O. aghardü (Norway) (SKULBERG and SKULBERG, 1985) showed marked similarity to the microcystin RR (arginine-arginine variant) (W. CARMICHAEL, Wright State University, Dayton, OH, personal communication) previously described for the toxic components of Microcystis aeruginosa (ERIK3SON et al., 19886). At present, O. aghardü is the only species among freshwater Oscillatoriaceae to have been chemically investigated for toxic blooms (SKULBERG et al., 1984; MERILUOTO et al., 1989). In Italy, toxic blooms of Oscillatoria rubescens were first reported in 1985, in a reservoir system used in Sardinia for the production of drinking water (VOLTERRA et al., 1985; Lolzzo et al., 1988). Following this report, we detected other toxic blooms of the genus Oscillatoria throughout the country (unpublished observations) . The present work is based on the chemical identification of microcystin-like toxins in toxic blooms of various Oscillatoria species, other than O. aghardü, which have been found in the Italian regions of Sardinia and Lazio . Author to whom correspondence should be addressed . 1307
Short Communications
1308
TeeLE 1. Toxcc veLVE (EC,~ oN MICROTOx) eND Toxuv ooNrrffvr (oN HPLC) oF THE THREE aLOOt~rs FROe~ rHE ITALIAN LAEF3 AND OF THE 9TRAIN OF OOMPARLION
Sample/species
Lake/year
TOxin COAient (m8/g dry cells)
ECm
(mg dry cells/ml) -LR like
(1) O . aghardü CYA hTIVA 38 (2) O . teraris +O . r'ubescens (3) O. terrwrs +O. nwugetit
+O.
rubescercs
(4) O . rubeacens
-RR like
-YR like
(Gjersjon, 198~
0 .28
-
2 .90
-
(Simbirizzi, 1989)
2.12
-
0 .48
-
(Flumendosa, 1989) (San Puoto, 1990)
1 .91 0.40
-
-
0.22
1 .16
See text .
The following blooms were examined : (1) mixed blooms of Oscillatoria tennis and O. rubescens (6 .5 x 10° and 7 x 10~ trichomes/litre, respectively, Lake Simbirizzi, January 1989); (2) mixed bloom of O. tennis, O. mougetü and O. rubescens (with a dominance of the species Oscillatoria tennis, Lake Flumendosa, September 1989); (3) O. rubescens bloom (Lake San Puoto, Lazio region), where this species represented 99% of the entire biomass, March 1990. Toxicity tests were performed using lyophilized algae. After extraction of 10 to 200 mg of material (MERILUl7T0 and ERIKS80N, 1988), the samples were tested with a Microtox .d., Microtox Application Notes: system (Microtox Operation Book, Microbics Inc. F M 103-116; U.S. Environmental Protection Agency, 1979). The results were processed statistically on a computer to give the sc~ 5 min value (extinction coefficient of 50% of light emission, after 5 min of exposure to the toxic substance) . The aplysiatoxins test (extraction according to MYND1~tSSE et al., 1977) was performed on female nude mice HRS/J and New Zealand rabbits, as per WHO (Organisation Mondial de la Santè, 1986); experiments were carried out according to the institutional laws on humane care of the animals . Drops of various quantities of the extract (10 ~l for mice and 10 ml for rabbits) were placed on shaven portions of the dorsal skin, and subsequently the animals were checked for cutaneous rashes for an entire week . The chemical identification of toxins was carried out by an HPLC Beckman System Gold instrument, using a Regis column ISRP GFF SS-80 (MERII,UOTO and ERIxssolv, 1988) and a C18 RP Lichrochart column (ERIKSSON et al., 19886), using a mobile phase of 12% acetonitrile/buffer 0.1 M potassium dihydrogen phosphate, pH 6 .8, flow rate 1 mlJmin, wavelength 238 nm with 0.005 AUFS and sample volume loaded 50 pl . Results shown here refer to the Regis column, which separates the RR, YR and LR microcystins used as standards (M131tII,UOTO et al., 1990). The microcystin standards were very kindly provided by Dr M. WATANABB (Metropolitan Research Lab. of Public Health, Tokyo, Japan - -YR tyrosine-arginine variant and -RR arginine~arginine variant microcystins) and Dr W. CARMICHAEL (Wright State University, Dayton, OH, U.S.A . - -LR leucine-arginine variant microcystin) . The samples of the lyophilized strain of O. aghardü ~CYA NIVA 38 (derived from Lake Gjersjon, Norway), were generously donated by Dr J. Eltlxssox (Ä6o Academi, Finland), and were primarily used as comparison for qualitative and quantitative measurements of the toxic peak of the HPLC .
Short Communications
m cuc a ô aN Q
1309
u c
0 a ô
0
S
Time
T
10
15
(min)
(E)
0
S
,
Tlme
10
(mln)
-YR
-LR 0 u C O
a O
aN a
-RR
5
Tlme
(mln )
FiCi . 1 . Ct~tOMA1'OßRAPHIC ANALY~9. OsciUatoria gghardü (red var .) CYA NIVA 38 strain (Lake Gjersjon, Norway) ; (H) O . rubescerts, O. teraris (Lake Simbirim, Sardinia ngion), responsive for -RR atendard; (C) O . rubescens (red var.) (Lake San Puoto, Lazio region); (D) O . rubescens, O . tertuis, O. »tmgetü (Lake Medio F7umendosa, Sardinia region), responsive for -YR standard, compared with the profile of standard toxins . (H) Comparative HPLC profile for standard toxins. Arrows iadicate the toxic peaks . (A)
15
1310
Short Communications
All of the Oscillatoria rubescens algal blooms, tested by the Microtox system, showed values ranging from 0.40 mg/ml (O . rubescens from Lake S. Puoto) to 2.12 mg/ml (O. rubescens from Lake Simbirizzi) (Table 1). The HPLC analyses showed an RR-like toxin for O. tennis plus O. rubescens blooms from Lake Simbirizzi, and a YR-like toxin for O. rubescens from Lazio region as well as from the mined population of O. temtis, O. mougetü and O. rubescens from Sardinia (Table 1, Fig. 1). A previous extract of Oscillatoria rubescens collected from Lakes Mulargia and Flumendosa, in Sardinia, during 1985, and stored at -80°C, gave the same RR-like peak as the bloom tested from Lake Simbirizzi (data not shown). Because of the repeated appearance of non-toxic blooms of Oscillatoria tennis in Lake Simbirizzi (four cases were examined from 1989 to 1990) and because of the presence of an RR-like toxin in a quasi pure bloom of O. rubescens (1985) from Lake Mulargia, the presence of a YR-like toxin in another bloom from Lake Mulargia in September 1989 is thought to be due to the O. mougetü component. Tests performed to verify whether the observed blooms of Oscillatoria could be responsible for cutaneous rashes similar to those caused by aplysiatoxin were negative (MOORS, 1982). Previous chemical studies of toxic Oscillatoriaceae have used the species O. aghardü. In its chemical characterization, the main toxic principle was microcystin-RR (ERIKSSON et al., 19ß8b; MERILUOTO et al., 1989), and therefore this kind of toxin is expected also in other species of Oscillatoria . However, the extract of the bloom of O. rubescens (red var.) from Lake S. Puoto (Lazio region) in March 1990 revealed the presence of a YR-like toxin, and the same was reported for a bloom of Oscillatoria dominated by the species O. tennis, O. mougetü and O. rubescens from Lake Fhimendosa (Sardinia) during September l9ß9 . The presence of hepatotoxic microcystins in various freshwater species of the genus Oscillatoria is not just an ecological problem (ERIICSSON et al., 1986; LINDHOLM et al., 1988). When waters are used for drinking purposes (as in the case of Simbirizzi-Fhurlendosa) it becomes a human health problem. During the potabilization process, Oscillatoria trichomes may be disrupted, and their hepatotoxins released into the drinking water (VOLTERRA et al., 1990). Notwithstanding the taxonomic differences existing between Mycrocystis and Oscillatoria, the production of similar toxins in the two genera raises interesting questions about the evolution of genetic systems showing the same biosynthetic pathway. Ec~
REFERENCES
Besa, K., Setn.eaea, O. M., Sxur aHta, R., UxnESnN., B. and Wn.irrv, T. (1986) Observations of toxic bluo-green algae (Cyanobactetia) in some Scandinavian lakes. Acta Vet. Stand. 27, 440-452. Bma, K., Wnutv, J., C~cxwer., W. and Deaxot .tun, A. (1988) Isolated rat liver perfusion studies with cyclic heptapeptide toxins of Microcystis and Oscillatoria (freshwater cyanobacteria) . Toxicon 26, 827-837. Esncssox, J. E., Mtoeu.uoro, J. A. O. and LQroxot.st, T. (1986) Can cyanobaoterial peptide toxins accumulate in aquatic food chains? Pros. 4th Int. Symp. Microbial Ecol. Ljubljana (Yugoslavia), pp. 655-658 (Meaus~a, F. and Guv'r~tt, M., Eds). Ljubljana: Slovene Society for Microbiology Press. ErutcssoN, J. E., Mettu.uaro, J. A. O., Kurnw, H. P., Jeep. Ar.-LeYr.K, K. and CODD, G. A. (19ß8a) Cellular effects of cyanobacterial peptide toxins . Toxicity Assess. 3, 511-517 . Esurssox . J. E., Mmur.utmo, J. A. O., KUS~w, H. P. and Stctn.r~c, O. M. (198ßb) A comparison of toxins isolated from the cyanobacteria OsciUatoria aghardii and Mlcrceystis aerugtnosa. Comp . Biothem. Physiol. ByIC, 207-210. Lu~mt~tor.x, T., Exnss9ox, J. E. and MESn utrro, J. A. O. (1988) Toxic cyanobacteria and water quality problems . Examples from a eutrophic lake on Aland, SW Finland. Woer Res. 23, 481~ß6. Lotzzo, A., SscFn, N., Vor r~ew+, L. and Cormr, A. (1988) Some features of a bloom of Oscillatoria rulxscens D.C. registered in two Italian reservoirs . Water, Air, SoA Poll. 38, 263-271.
Short Communications
131 1
Mtatn,uoro, J. A. O. and Eturssox, J. E. (1988) Rapid analysis of cyaaobacterial peptide toxine . J. Chromas . 4~, 93-99. Mta