博士 / 國立臺灣大學 / 動物學系 / 86 / ABSTRACT
In Taiwan Microcystis blooms are frequently found in
reservoirs and in the fresh or brackish aquaculture ponds. In
order to realize the adverse effects these Microcystis strains
may exert, we started a series of surveys including water
sample collection, cell isolation, and toxin analysis of the
blue-green algal blooms five years ago. In this research
microcystins were isolated from the cultured strains of M.
aeruginosa and a scum mass of a blooming Microcystis.
Toxin profiles were compared among cultured strains of M.
aeruginosa and the field sample by high performance liquid
chromatography. In addition, both the mouse and brine
shrimp toxicity assay were applied in the relative toxicity
analysis of different microcystins and M. aeruginosa strains.
Extract of a scum mass of Microcystis spp. collected from
an eel pond at Duujia, Tainan in September of 1994 showed a
threshold toxicity of an amount between 50 and 250mg dry
alga per kg mouse weight in the mouse bioassay. It was
noted that the death response occurred usually 40min after the
i.p. injection of the lethal dosage of cell extracts. It
appeared that at least 40min were required to show the death
response after i.p. administration of the toxin extracts, and
then, diffusing into the bloodstream, arriving at the target
organ and exerting the toxic functions till death. Autopsy
showed the mice were died from hypovolaemic shock due to
the hepatic haemorrhage. All the poisoned mice were
observed to have dark-red and swollen blood-engorged livers,
a typical symptom of microcystin poisoning, after dissection
examination. Brine shrimp, Artemia salina toxicity assay
was applied and compared with the mouse bioassay in the
screening of toxic strains among the nine blue-green algal
isolates in this study. It was found that M.TY-1, M.TY-2,
M.CY-1, M.TN-2, M.TN-3, and M.TN-4 strains of M.
aeruginosa were toxic, consistently by either of these two
bioassays. The same conclusion was also drawn by the
HPLC analysis of known microcystins in the extract of
different algal strains. It seems that the brine shrimp
toxicity assay can be a replace to the mouse assay in screening
of the toxic Microcystis strains due to its inexpensiveness and
convenience in operating the tests.
Ten toxic components were isolated from cell masses of
the cultured strain, M.TN-2 and the field sample by a series of
separation on a Sephadex LH-20 column, a Si-flash column
and a reverse phase high performance liquid chromatography.
These compounds were identified as MCYST-LR (1), -YR (2),
-FR (5), -WR (6), -RA (9), -RR (10) in MCYST group,
[Dha]MCYST-LR (3), -RR (4) in [Dha]MCYST group, and
[D-Asp]MCYST-FR (7), -WR (8) in [D-Asp]MCYST group by
comparing their 1H-NMR, and COSY-45, 1H-1H COSY,
TOCSY, DEPT, HMQC and HMBC NMR analysis with that of
the known authentic MCYST-LR in addition to the molecular
weights determined by MALDI-TOF mass spectrmetry.
Amino acid compositions of these microcystins were further
analyzed by the HPLC separation on the Dabsyl derivatives of
their acid hydrolysates. The chirality of some of the
constituent amino acids was determined by the GC-MS
analysis of the PFP-IPA derivatives following acid hydrolysis
of these toxins. Among the ten microcystins isolated, [D-
Asp]MCYST-FR, -WR and MCYST-RA were found new to the
group of published microcystins. Nuclear magnetic
resonance studies of the MCYST-LR showed an up-field shift
of the 1H chemical shifts of MeAsp H-2 and Glu H-2 and a
down-field shift of Adda H-2 as the toxin was deprotonated.
This observation suggested a conformational change of the
cyclic heptapeptidyl structure occurred while the toxin were
placed in a basic solution.
Some of the purified microcystins showed their LC50 within the
range of 12.7 and 22.3g/ml on the brine shrimp. The
potency of brine shrimp toxicity of these
microcystins was found to be MCYST-FR MCYST-WR
MCYST-RR MCYST-RA > [Dha]MCYST-RR >
MCYST-LR. This result was noticed opposite to the finding that
MCYST-LR was the most toxic microcystin known to mice in
publications. A commercial available ELISA kit with monoclonal
antibodies against MCYST-LR was tested to observe the cross-
reactivity of the purified microcystins in this research. It
was found that all the tested microcystins, MCYST-FR, -WR,
-RR, -RA, and [Dha]MCYST-RR, -LR, and [D-
Asp]MCYST-FR, -WR showed cross- reactivity
lower than 50% except MCYST-RR. MCYST-RA, [D-
Asp]MCYST-FR and [D-Asp]MCYST-WR showed cross-reactivity
even lower than 20% by this ELISA kit. It is suggested that the
quantity of microcystins may be underestimated by using this
ELISA kit in the analysis due to the lower cross-
reactivity of microcystins with a different amino acid
substitution at the second or the fourth position or
with a demethylated analogue at the third position.
| Identifer | oai:union.ndltd.org:TW/086NTU00312001 |
| Date | January 1998 |
| Creators | lee, Tzong-Huei, 李宗徽 |
| Contributors | Hong-Nong Chou, Chen Hong-Cheng, 周宏農, 陳弘成 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 183 |
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