REPAIR AND MUTAGENESIS IN PROKARYOTS AND EUKARYOTS
Main researcher
: FRANEKIĆ, JASNA (85870) Assistants
SKUPNJAK, ŠTEFICA (151373)
MATIJAŠEVIĆ, ZDENKA (52033)
BAŠIĆ-ZANINOVIĆ, TAMARA (130265)
BAČUN-DRUŽINA, VIŠNJA (107745)
PETAK-NEKIĆ, KATICA (182243)
TOMIČIĆ, MAJA (900693)
Type of research: basic Duration from: 03/01/91. to 03/01/95. Papers on project (total): 63
Papers on project quoted in Current Contents: 23
Institution name: Prehrambeno-biotehnološki fakultet, Zagreb (58) Department/Institute: Department of Biochemical Engineering Laboratory of Biology and Microbial Genetics Address: Pierottijeva 6 City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (01) 417-044
Fax: 385 (01) 411-436
E-mail: jfranek mapbf.pbfrng.hr
Summary: The genotoxic effects of different classes of chemicals
(model mutagens, non-ionizing radiation, pesticides, drugs and their
metabolites) were investigated both in vitro and in vivo. The obtained
results support the hypothesis that some carcinogens (ETU) are capable of
disturbing the chromosomes segregation through their interaction with
non-DNA targets. The aim of this study was to validate the short-term
test-system with yeast for the detection of substances capable to induce
aneuploidy. Our study has shown that human tumour cells resistant to
specific alkylating chemotherapeutic drugs had higher 3-methyladenine DNA
glycosilase activity comparing to normal cells. By using shuttle vector
methodology and by analyzing mutational spectra induced by potent
carcinogen MNU, we investigated the influence of different DNA
polymerases involved in the replication process, on the fidelity of
replication of DNA strands (in one case leading and in the other lagging
one) opposite damage caused by MNU, and therefore, the effects on the
mutation frequency and distribution of mutations. The results have shown
that different DNA polymerases had no influence on two studied
phenomenons. Investigation effects of transcription on UV-induced
mutations in mammalian cells showed a clear bias towards the transcribed
strand under both basal and induced gpt transcription. The results have
shown that in S.typhimurium nucleotide excision repair (NER) is the main
DNA repair system operating on cytotoxic and premutagenic DNA lesions
induced by monofunctional alkylating agents (MNNG, MMS, ENNG, ENU, PNNG
and BNNG). On the chromosomal DNA of S.typhimurium we have not detected
nucleotide sequence that could hybridize with alkA gene from E.coli. The
aidB gene expression was induced in E.coli (strain MV1161) and in
S.typhimurium (strain G46/pMV201) by two studied alkylating agents, MMS
and MNNG. The genotoxic effect of pulsed and pulsed color Doppler
ultrasound then continuous and pulsed microwave radiation were
investigated with microbial test systems for detecting mutation
recombination and aneuploidy. Pulsed and pulsed color doppler (intensity
0.59 W/cm2 and frequency 5 MHz) showed the mutagenic activity in strains
TA100 and TA98 of bacteria Salmonella typhimurium. Continuous and
pulsed microwave radiation power density 16 mW/cm2 and frequency 7.7 GHz
caused weak mutagenic effects in strains TA98 and TA1538. Both types of
non-ionizing radiation did not induce recombination and aneuploidy during
mitosis in yeast Saccharomyces cerevisiae.
Research goals: Biological monitoring consists of detecting,
characterizing and evaluating the effects produced in individuals exposed
to potential mutagens and/or carcinogens. Pesticides have become a major
source of environmental mutagens. Amongst the pesticides tested in vitro
and in vivo, many of them as well as their metabolites have been shown
mutagenic properties. We have been investigated model chemicals for their
ability to induce aneuploidy in yeast Saccharomyces cerevisiae and mound
Aspergillus nidulans. We have used a cellular test systems to study the
possible correlation between induction of aneuploidy on the onehand and, on
the other, inhibition of polymerization mammalian brain tubulin in vitro.
Extract of glial cell line (SF-126) which is sensitive to the cytotoxic
effect of the haloethylnitrosoureas and cell line (SF-188) which is
resistant to these agents have been tested for their ability to release
methylated bases from DNA substrate which has been modified with (3H)
dimethyl sulphate. In comparatione with the sensitive cell line, extracts
from the resistant cell line have 2-3 fold higher enzymatic activity. It is
known that one of the sources of mutations may be the replication process
itself because of the different fidelity of DNA polymerases conducting the
synthesis of leading and lagging strand opposite the damage (in our case an
alkylation damage). The use of two "shuttle" vectors with the target gene
in opposite orientation relative to the origin of replication give us the
opportunity to study the influence of different DNA polymerases on mutation
frequency and their distribution. The results have shown that DNA
polymerases had no effects on studied phenomenons. According to the program
of research we have investigated the low dose effects by monofunctional
alkylating agents: MNNG, MNS, ENNG, ENU, PNNG and BNNG. Comparing the
S.typhimurium uvrB mutant to nucleotid excision repair-proficient strain it
has been noticed that the latter was mutagenized more efficiently at low
doses and less efficiently at high doses of methylating and ethylating
agents. The starting point of this work was disagreement of scientists
about the existence of quantifiable injurious effects of doppler
ultrasound and microwave radiation on biological systems. The mutagenic
effects of these types of radiation were determined by employing a battery
of tests (Ames test) Salmonella typhimurium (strains TA100, TA98, TA97,
TA1535, TA1538) and the yeast Saccharomyces cerevisiae (strains D7,
D61.M). Plate incorporation assay with S. typhimurium demonstrated direct
mutagenicity of pulsed and pulsed color doppler ultrasound 0.59 W/cm2
spatial peak temporal average intensity (SPTA) and frequency 5 MHz.
Continous and pulsed microwave radiation power density 16 mW/cm2 and
frequency 7. GHz causes a weak mutagenic effect in strains TA98 and TA1538
for detecting frameshift mutations. Pulsed and pulsed collor doppler and
continuous and pulsed microwave radiation did not induce aneuploidy
(strain D61.M) and recombination (strain D7) during mitosis in yeast S.
cerevisiae.
COOPERATION - PROJECTS
Name of project
: EZ/COST 66 Fate of pesticides in the soil and
environment Name of institution: Commission of the European Communities City: Brisel, Belgija
COOPERATION - INSTITUTIONS
Name of institution
: Istituto Superiore di Sanita, Laboratory of
Comparative Toxicology and Ecotoxicology Type of institution: University/Faculty Type of cooperation: Joint publishing of scientific papers City: Rim, Italija
Name of institution
: Department of Molecular Genetics and
Microbiology, University of Massachusetts, Medical School, Worcester, Type of institution: Economical/Production Type of cooperation: Joint publishing of scientific papers City: Massachusetts, USA Other information about the project.