Environmental and Molecular Mutagenesis - Legacy Tobacco ...

38 1989 EMS Abstracts
Notes The RAD4 gene requiied for incision of UV-induced excision repair in Saccharomyces
cerevtsiae has been isolated by phenotypic complementation of a UV-sensitive rad4-4
mutant with yeast genomic library amplified In E .coli (Yoon et al .,1985) . The yeast
insert in recombinant plasmid designated as pPCl was isolated in the 2 .6Kb BglII-gamHI
fragment in the aubcloned pPC100 and contained a functional RAD4, but not a suppressor
tRNA gene as determined by restriction mapping and DNA-tRNA hybridization . In addition,
the observation that all different plasmids harboring yeast insert conferred UV
resistance to rad4-4 cells to a similar level regardless of their copy number, strongly
suggests that our cloned gene does not contain a suppressor . Northern blot analysis
with pPC100 as the probe indicates that the insert DNA containing the entire RAD4 gene
can hybridize with transcripts of 1 .2Kb and 2 .3Kb in length . Stationary-phase cultures
of E .co1i BL21(DE3) strain transformed with plasmids harboring the cloned RAD4 gene
showed a considerable delay in the resumption of exponential growth and a dramatic
reduction in the production of host proteins . Moreover, the overexpressed Rad4 protein
estimated as 89Kd by 2-D gel analysis revealed a toxic effect In Z .coli, suggesting
that the Rad4 protein interferes with normal growth control in 6 .co1t cells .
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USE OF THE BALB/c-3T3 ACAR SUSPENSION ASSAY TO DETECT CHEMICAL-INDUCED TRANSFORMATION .
M . A . Cifone, L . Custer and E .J . Matthews, Hazleton Laboratories America, Inc .,
Kensington, Maryland .
An alternate endpoint to morphological transformation In the BALB/c-3T3 transformation
assay has been described . In this assay, the formation of colonies in agar was
measured after chemical treatment . Approximately 50,000 cells were seeded on day 0 and
treated on day 1 with a chemical for 24 hours . The cells were then kept in logarithmic
phase for 11 to 13 days and plated in 0 .4% Noble agar . Four weeks later the colonies
were fixed with 2 .5% glutaraldehyde and colonies with diameters greater than 0 .1 mm were
automatically counted using an Olympus Q2 1mage analyzer . Fifteen chemicals were
investigated In this system and activities were compared to the presence or absence of
structural alerts, and published results obtained in the standard BALB/c-3T3
transformation assay, several in vitro genotoxicity assays, and rodent bioassay . The
chemicals studied included four genotoxic carcinogens, six genotoxic nonearcinogens and
five nongenotoxic carcinogens . The results with the agar suspension assay closely
correlated with the results obtained with the standard BALBe/-3T3 transformation assay .
However, in some cases the agar suspension assay appeared to be les∎ sensitive . Some
chemicals gave an equivocal or negative response In the agar suspension assay and ware
weakly active in the standard transformation assay . While the BALB/c-3T3 agar
suspension assay is labor intensive and not appropriate for routine screening, it
measures an endpoint that is crucial to the transformation process and can be used to
answer mechanistic questions .
(supported by NIEHS Contract No . N01-ES-65150) .
PARALLEL DETECTION OF SCE, DNA ADDUCTS AND PROTEIN ADDUCTS IN MICE DOSED WITH THE
CARCINOGEN 2-ACETYLAMINOFLUORENE .
106
G .Citro, R .Zito, A .Verdina, R .Galati, R .Benigni, P .Leopardi, A .Zijno, and R .Crebelli,
Istituto Superiore di SanitB and Istituto Regina Elena, Rome (Italy) .
In order to explore the interrelationships among DNA adducts, protein adducts and
SCE simultaneously induced in the complexity of in vivo situation, an investigation
was carried out in Swiss mice treated with the carcinogen 2-acetylaminofluorene (2AAF) .
Groups of 4 male mice were treated either by gastric intubation (at 25,50 and 100 mg/kg
on 4 consecutive days) or by chronic dietary exposure (2 months feeding with a satura-
ted 2AAF water solution) . For each animal the frequency of SCE in bone marrow cells,
as well as the levels of binding of 2AAF to liver and spleen DNA and blood proteins we-
re determined by means of cytogenetic techniques and competitive immunoassays, respect .
The results obtained highlight a dose-related trend in SCE rates after acute exposure,
with wide interindividual variance wich was significantly related to individual levels
of liver DNA adducta . Conversely, the amount of covalent binding to spleen DNA proved
to be related only to individual levels of binding to blood proteins . Such picture was
modulated by the schedule of treatment : chronic 2AAF exposure in fact produced relati-
vely higher levels of binding to spleen than to liver DNA (about 1,000 and 500 adducts/
million nucleotides, respect .) and did not increase significantly SCE rates in bone
marrow .
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