162 Chem. Res. Toxicol., Vol. 11, No. 3, 1998
Communications
Ch a r t 2. Str u ctu r es of Micr ocystin -LR,
[4(Z)-Ad d a 5]m icr ocstin -LR, a n d
[6(Z)-Ad d a 5]m icr ocystin -LRa
Sch em e 1. P ossible Ou tlin e for Br ea k d ow n of
Micr ocystin -LR by UV Ir r a d ia tion
a
The conjugated dienes in the Adda moieties of microcystin-
LR, [4(Z)-Adda5]microcystin-LR, and [6(Z)-Adda5]microcystin-LR
are 4(E),6(E), 4(Z),6(E), and 4(E),6(Z), respectively.
metrical isomers from microcystin-LR by UV irradiation.
The decomposed compounds eluted near the solvent front
by HPLC.
Ta ble 3. Recover y a n d Com p osition of P h otor ea ction
P r od u cts (Rea ction con d ition s d escr ibed in th e text)a
In the case of [6(Z)-Adda5]microcystin-LR (0.1 mg/10
mL), only 28.3% of the original was lost by UV light for
15 min, and the isomerized compounds from [6(Z)Adda5]-
microcystin-LR were [4(Z)-Adda5]microcystin-LR (15.0%),
[6(Z)-Adda5]microcystin-LR (15.1%), microcystin-LR
(68.6%), and [tricyclo-Adda5]microcystin-LR (1.3%). These
results were almost the same as those of microcystin-
LR and [4(Z)-Adda5]microcystin-LR.
These results and the configuration of [tricyclo-Adda5]-
microcystin-LR show that [tricyclo-Adda5]microcystin-LR
was formed from the original microcystin-LR reversibly
and was unstable under UV.
composition (%) of
recovered products
ratio
reaction recovery
time (min)
(%)
LR 4(Z) 6(Z) tricyclo 4(Z)/LR 6(Z)/LR
0
15
30
45
60
75
100
100
0
0
0
71.5
64.6
58.9
53.0
50.2
67.4 15.0 14.9
64.9 15.1 15.3
64.5 14.9 14.9
64.4 14.8 14.4
63.9 14.4 14.6
2.7
4.7
5.7
6.4
7.1
0.22
0.23
0.23
0.23
0.23
0.22
0.24
0.23
0.22
0.23
a
LR, microcystin-LR; 4(Z), [4(Z)-Adda5]microcystin-LR; 6(Z),
[6(Z)-Adda5]microcystin-LR; tricyclo, [tricyclo-Adda5]microcystin-
LR.
In order to determine the LD50 of the isomers of
microcystin-LR in mice, purified [4(Z)-Adda5]-, [6(Z)-
Adda5]-, and [tricyclo-Adda5]microcystin-LR and micro-
cystin-LR were injected into the peritoneum of the mice.
When 100 µg/kg of mouse of microcystin-LR was injected,
all mice in the group died. However, no mouse in each
group died when 1500 µg/kg of mouse of [4(Z)-Adda5]-,
[6(Z)-Adda5]-, or [tricyclo-Adda5]microcystin-LR was in-
jected. These results suggest that 4(E),6(E)-Adda is the
essential structure for hepatotoxicity.
The results of recoveries and compositions of the
products during the reaction are summarized in Table
3. The recoveries of the three major compounds and
unreacted microcystin-LR decreased gradually during the
reaction. About 50% of microcystin-LR was decomposed
by UV irradiation for 75 min (0.585 J ). In this reaction
condition, [4(Z)-Adda5]- and [6(Z)-Adda5]microcystin-LR
were given in about equal amount, and the ratios of [4(Z)-
Adda5]microcystin-LR/microcystin-LR and [6(Z)-Adda5]-
microcystin-LR/microcystin-LR were constant during the
reaction. These results demonstrate that the reaction
from microcystin-LR to [4(Z)-Adda5]microcystin-LR and
[6(Z)-Adda5]microcystin-LR was reversible and in equi-
librium. On the other hand, the relative content of
[tricyclo-Adda5]microcystin-LR in the recovered products
increased gradually.
Discu ssion
Formation of [6(Z)-Adda5]- and [4(Z)-Adda5]microcystin-
LR as geometrical isomers of the Adda moiety of micro-
cystin-LR was reversible. The equilibrium constants of
the two isomer formations were almost the same.
The formation of [tricyclo-Adda5]microcystin-LR sug-
gested that the [2 + 2] addition between the double bond
at position 6-7 and the double bond at the quaternary
carbon-ortho position of the phenyl group was performed
under UV light. Previously, a three-dimensional struc-
ture of Adda in microcystin-LR has been examined using
computer modeling by Lanaras et al. (9). They showed
that the Adda formed a “U-shape” and the phenyl group
located around the conjugated diene. In order to confirm
the three-dimensional structure of Adda of microcystin-
LR in the aqueous solution, ROE (rotating Overhauser
effect) was examined in D2O. In the experiments, ROE
between H-5 at the conjugated diene and H-17 as a pro-
ton at the meta position of the phenyl group was ob-
served. These results support that the Adda moiety of
microcystin-LR formed a “U-shape” in aqueous solutions.
The U-shape of the Adda moiety in aqueous solutions is
suitable to form [tricyclo-Adda5]microcystin-LR.
[Tricyclo-Adda5]microcystin-LR was formed from mi-
crocystin-LR reversibly and was decomposed under UV.
These facts suggest that the breakdown of microcystin-
LR proceeds via [tricyclo-Adda5]microcystin-LR (Scheme
1), the cyclization velocity is faster than that of the
breakdown, and the ring-opening velocity from [tricyclo-
Adda5]microcystin-LR to microcystin-LR is slower than
that of the cyclization or the breakdown.
When the aqueous solution of [tricyclo-Adda5]micro-
cystin-LR (0.1 mg/10 mL) was placed under UV light
for 15 min, 68.3% of [tricyclo-Adda5]microcystin-LR de-
composed, and the remaining compounds were [4(Z)-
Adda5]microcystin-LR (3.1%), [6(Z)-Adda5]microcystin-
LR (3.1%), microcystin-LR (14.3%), and [tricyclo-Adda5]-
microcystin-LR (11.2%). The ratios of [4(Z)-Adda5]micro-
cystin-LR/microcystin-LR and [4(Z)-Adda5]microcystin-
LR/microcystin-LR agreed well with those of the geo-
Our results suggest that detoxification of microcystins
can be performed by UV irradiation at 239 nm as the
max of microcystins due to the conjugated diene in Adda.
λ