Environmental and Molecular Mutagenesis - Legacy Tobacco ...

20 1989 EMS Abstracts
Notes URINARY BIOMARKERS 0F ENDOGENOUS DNA DAMAC+E
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
D .S . Bergtold and M .G . Simic
National Institute of Standards and Te chnoloNy, Gaithersburg, MD 20899
The biological consequences of damage to DNA by exogenous agents have been focal points
of enormous numbers of studies. Damage to DNA by andoRenous processes, in contrast,
has been investigated to a much smaller extent due to intrinsic difficulties, e .q .,
lack of controls, low levels of damage, lack of suitable biomarkera, etc. Using gas
chromatography/mass spectroscopy, we have developed absolute measurements of oxidative
urinary biomarkers based on the original work of Ames and coworkers. The urinary
levels of products (thymine glycol, 8-hydroxyguanine, their nucleoside moieties, and
others) have been correlated in different species to their metabolic rates, longevity,
and size . Since many of the observed urinary biomarkers are identical to •OH radical
reaction products with DNA bases, gamma irradiation has been used to elucidate the
underlying mechanisms by which metabolic biomsrkers eriae .
L .F .Bernini, A .T .Nstarajan . A .H .M .Schrauder-Rotteveel, P .C .Giordano, J .S .Ploem
and A .Tates .
Depart . of Human Genatics . Dept . of Radiation Genetics and Chemical Mutagenesis,
Dept . of Histochemistry and Cytochemistry, University of Leiden, Sylvius
Laboratoria, Wassenaarsavag 72, Leiden, The Netherlands .
ASSAY FOR SOMATIC MUTATIONS OF HUMAN HB}IOGLOBINS .
With the purpose of monitoring by cytoimmunocheaical methods somatic mutations
of globin genes, we have raised in rabbits monospecific polyclonal antisera
against a number of abnormal hemoglobins . Rare mutant calls are identified on
peripheral blood smears by indirect issmmofluorescence and counted by an
automatic scanning microscope especially constructed for the examination of Hb
abeorbance and FITC fluorescence . With the aid of a suitable adapter, glass
slides of 8x8 cm . containing about fifty million red cell acan be screened in
three hours . All fluorescent objects found on the smear undergo a computer
directed pattern recognition analysis . Only those objects having the size and the
shape of a red cell and absorbing at 413 nm are eventually classified as mutants
and checked through direct examination by an expariented observer . On the
average, five Hb S cells/10 • erythrocytes have been found in the peripheral
blood of healthy adult individuals . When, in the sam. person, three different
mutations are monitored simultaneously with a mixture of the relative antisera, a
correspondent increase of the frequency of mutant cells is observed . We report
and comment the results obtained with this assay in people exposed to genotoxic
agents or carriers of inherited abnormalities of DNA repair .
READTHROUGH OF CHEMICALLY INDUCED DAMAGES IN DNA DURING KLENOW FRAGMENT-MEDIATED
DNA SYNTHESIS .
Tadayoshi Beeshol, Kazuo Negishi2 and Hikoya Hayatsul
1Faculty of Pharmaceutical Sciences, 2Gene Research Center, Okayama University,
Tsushima, Okayama 700, Japan
In E . coli, DNA damages inducible by UV, X-ray irradiation and many chemical
carcinogens block DNA replication and exert "SOS response" . A characteristic event
accompanying this phenomenon is an enhanced mutation . A possible way to elevate the
mutation rate is "translesion" DNA synthesis ; but its mechanism remains unclear . We
have shown the direct role of RecA protein in such a translesion DNA synthesis, with
N4-amino-5,6-dihydrocytosine-6-sulfonate residues as the DNA base damage . This
modification can be formed on single stranded M13mp2 DNA by treatment with a bisulfitehydrazine
mixture . From the analysis of in vitro DNA chain elongation products using
sequencing gels, we were able to show that this damage can block DNA chain elongation
at one nucleotide before the damage . In the presanse of RecA protein, Rlenow fragment
was able to readthrough this damage ; but in the presense of dGMP, an inhibitor for
exonuclease activity, the DNA chain elongation remained to be blocked . A high rate of
dCMP/dCTP turnover during the translesion DNA synthesis was observed, as detected by
chromatographic analysis of the nucleotides in the reaction mixture . Therefore,
enhanced exonuclease activity and the binding of RecA protein to damaged DNA may affect
the processivity of the polymerase and this effect may be important in the translesion
DNA synthesis .
50
51
52
tn
m
CO
Oh
t0
W
tn W
N