Rapp. Comm. int. Mer Médit., 36,2001
193
Introduction
The estimation of genotoxic potential in the marine environment
can be carried out by measuring genetic endpoints which exhibit pri-
mary DNA damage such as strand breaks.The Fast Micromethod
®
measures changes in the pattern of DNA denaturation by directly alter-
ing DNA integrety in cell or tissue lysates at alkaline pH, and follow-
ing their time dependence (1).Two of the major advantages of the
method are small amount of sample (cells suspension or solid tissues)
needed and the short time of analyses.The aim of this study was to
establish the applicability of the Fast Micromethod
®
for its use in
marine ecotoxicological monitoring research.
Methods and materials
The Fast Micromethod
®
is performed according to Batel et al.(2).
The time course and the extent of DNA denaturation are followed
directly in the microplate by measuring the ?uorescence of the
D
NA/PicoGreen complex for 30m
n
.Results are calculated after
7-10 minutes of denaturations and expressed as strand scission factors
(SSFs) calculated as:
SSF = log
10
(% dsDNA
treated sample
/%dsDNA
control sample
)
Double-stranded DNA percentages were calculated in relation to
?uorescence values at 0-time denaturations after correction for blank
readings which represents relative 100% of ds-DNA before denatura-
tion in samples.Thus SSF=0 assumes absence of DNA strand breaks
and alkali labile sites, while SSF<0 indicates increasing frequencies of
strand breaks and alkali labile sites in samples.For pratical reasons the
SSF (strand scission factors) in graphical presentations were multi-
plied by (-1).
Results and discussions
The effect of 0-50µM Bleomycin-Fe(II) complex on the induction
of DNA strand brakes in Holothuria tubulosa (3567A) cells has shown
a dose dependent response with SSFx(-1) values from 0.047 to nega-
t
ive control to 0.537 respective
l
y.The dose-response histogram for
0 -500 rad
?
-irradiated spongeSuberites domuncula cells has shown
that average SSFx(-1) varied from 0.020 (control cells) to 0.082 for
500 rad (irradiated cells).
MusselsMytilus galloprovincialis, injected with 0-1 µg NQO/g
mussel, showed dose-dependent DNA damage with SSFx(-1) values
from 0.000 for negative control to 0.168 for 1µg NQO/g.The effect of
0-83.3 µMBleomycin-Fe(II) complex on the induction of DNA strand
breaks in mussel gills has shown SSFx(-1) values 0.000 (control mus-
sel) to 0.140 respectavely.
A different amount of DNA damage (SSFx(-1) values from 0.738 to
-0.610) from different locations along the Adriatic coast (Fig.1) was
detected during two years of observations (August 2000-June2000).In
the cases where histograms (SSFx(-1- values) were negative, it was
presumed, according to Vukmirovic (3), that the DNA-DNA and/or the
DNA-protein crosslinks are presented.
This study represents a suitable Fast Micromethod
®
for DNA dam-
age in native DNA isolated from cotton-spinner Holothuria tubulosa,
marine sponge cells Suberites domuncula and mussel gill (Mytilus
galloprovincialis) tissue.
The Fast Micromethod
®
is applicable for measurement of DNA
integrity of small samples for genotoxicity assessment in environmen-
tal monitoring for genotoxic effects of lower taxa or sesile organisms
(sponges, mussels).S
e n s i
t ivity and preciseness of the Fa s
t
Micromethod
®
is comparable with the widely used Comet assay (4)
which makes the former suitable for the assessment of pollution
impact on aquatic organisms that can be used as bio-indicators.
References
1. Muller W.E.G., Batel R., Zahn R.K., Bihari N. and Schroeder H.C.,
1
9
9
7
.Mikro-Methode zur Schnellbestimmung von DNA-Schäden und
deren Reparatur unter Verwedungs des Fluoreszenzfa
r
b
s
t
o
ffs PicoGreen
und ihre A
n
w
e
n
d
u
n
g
.D
e
u
t
che Patenteamt Muench
e
n, No.19724781.4-41.
2. Batel R., Jaksic Z., Bihari N., Hamer B., Fafandel M., Chauvin C.,
Schrode H.,C., Muller W.E.G. and Zahn R.K., 1999.AMicroplate Assay
for DNA Damage Determination (Fast Micromethod
®
) in Cell
Suspensions and Solid Tissues.Anal.Biochem.,270: 195-200.
3.Vukmirovic M., Bihari N., Zahn R.K., Muller W.E.G. and Batek R.
1994.DNAdamage in marine mussel Mytilus galloprovincialisas a
biomarker of
environmental
contamination,Mar.Ecol.
Prog. Ser., 109: 165-171.
4. Bihari N., Batel R.,
Jaksic Z., Muller W.E.G.,
Waldmann P.and Zahn
R.K.2001.Comparison
Between DNADamage
in HeLa Cells as
Measured by the Comet
Assay and Fast
Micromethod.
®
,Sci.Tot.
Env.in press.
Rapp. Comm. int. Mer Médit., 36,2001
DNA DAMAGE DETERMINATION IN GILLS OF THE MUSSEL MYTILUS GALLOPROVINCIALIS
L.(MOLLUSCA: BIVALVIA) BY FAST MICROMETHOD
®
Zeljko Jaksic
Laboratory for Marine Molecular Toxicology, Center for Marine Research Rovinj, Ruder Boskovic Institute,
Rovinj, Croatia - jaksic@cim.irb.hr
Abstract
Fast Micromethod
®
was applied in the determination of DNA damage as strand breaks alkali-labile sites and incomplete excision repair
in cell suspensions or tissue homogenates in single microplates.The chemical xenobiotics (4-nitroquinoline-N-oxide and blepmycine) and
different doses of
?
-rays, generated by Cs
127
, were used to induce DNA damage in native DNA and sponge cells.The method was used
in a monitoring program in the Adriatic Sea to detect the effect of mixed marine pollutanta.
Keywords : Adriatic Sea, bio-indicators, bivalves, ecotoxicology, monitoring
Fig. 1. - Results of SSF values for Mytilus galloprovincialisgill homogenates collected from different loca-
tions on the Adriatic coast during 2 years (August 1998 – June 2000).
