Environmental and Molecular Mutagenesis - Legacy Tobacco ...

29 1989 EMS Abstracts
TRANSFECTION WITH HUMAN STOMACH DNA AND BiCNU RESISTANCE OF CHO CELLS Notes
I .I . Arzimanoglou, C . Troungos, and S . Kyrtopoulos, N .H .R .F ., Athens 11635, Greece
The prem,tagenic, precarcinogenic alkylation lesion 06-alkylguanine is repaired
in repair-proficient cells by the enzyme O6-alkylguanine-DNA-alkyltranaferase (06-AGT),
an enzyme which in E .coli can be induced under conditions known as "adaptation" . The
phenomenon of adaptation has not been clearly demonstrated in eucaryotic cells . We
have exposed CHO cells (with very low 06-AGT activity) to low concentrations of ?RiNG
and then tested them for 06-AGT induction . No such induction was achieved . However,
following such pretreatment, a reduction in SCEs induction by a challenge dose of NNU
was observed, suggesting the induction of protective mechanism other than 06-AGT . In
an attempt to examine the expression of an eucaryotic ACT gene, we co-transfected
CHO cells with human DNA, and the bacterial neomycin gene, which is enclosed in
Cosmid Homer-6 . Control cells were transfected only with the latter .G418-rasistant
colonies were continuously cultured for a number of cycles of further selection with
the cross-linking, cytostatic, alkylating agent BiCNU (Carmustine) . 06-AGT has been
demonstrated to confer resistance, against the toxic effects of the cytostatic drug
BiCNU . The main results of the study are the following : 1) Following selection with
BiCNU, colonies with increased amounts of AGT were obtained from both transfected
and control cells, 2) While the average increase in 06-AGT levels was roughly the same
in colonies derived from transfected and control cells, larger number of BiCNU -
resistant colonies were obtained from transfected cells, 3) Cells containing increased
06-ACT, showed increased resistance against BiCNU and decreased mutability (to thioguanine-resistance)
by HNNG .
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FUTURE DEVELOPI[ENY OF SHORT TERM TESTS
J . Ashby . ICI Central Toxicology Laboratory, Alderley Park, Cheshire .
Two models of chemically-induced rodent carcinogenicity are currently competing for
attention . The first requires that all carcinogens induce cancer by virtue of their
assumed ability to modify directly DNA structure or functlon, a property that
presumably can be determ3ned in vitro given appropriate assays . The second model
recognizes the potential impoTr7Rf~'Zff biologlcal disturbances lnduced in rodents by
the administration of chemicals . These disturbances may be due to the chemical's
overt toxicity or to its ability to induce subtle changes in tissue hoaeostasis and
gene expression . If the chemical administration is chronic the associated biological
changes may assume pseudo- permanence and lead to modification of tumor lncidences in
the treated animal at death, in particular to 'carcinogenic' increases . If the
chemical is also genotoxic, then truly irreversible changes in gane expression .ay
accompany the temporal changes, and this could lead to more overt carcinogenic
consequences . This second model fits the available facts of rodent carcinogenicity
better than does the first model, however, the first model is sufficlent to account
for the large majority of trans-species and multiple site carcinogens, ie, those
carcinogens generally considered most likely to pose a hazard to humans are genotoxie
in vitro . Progress in this field will depend upon which of these two models is
accep e for development . If the goal is to detect DNA modifying carcinogens, such as
benzpyrene, then the only need !s to organize and refine current techniques and
testinh strategies . In contrast, if the requirement is to predict all future rodent
carcinogens, then other disciplines must be invoked to understand the nature and
significance of the many non-specific toxicities elicited by chemicals in rodents .
From such studies may emerge useful predictive assays for non-genotoxic rodent
carcinokens such as saccharin and limonene .
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THE SPONTANEOUS MUTATION SPECTRUM IN H13mp2 PHAGE
Gen-ichi Atsumll, Keiko Matsumotol, Tadayoshi Besshol, Kazuo Negishi2
and Hikoya Hayatsul
1 Faculty of Pharmaceutical Sciences . Okayama University, Tsushima, Okayama 700
2 Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan
Naturally occurring mutations include almost all conceivable changes in DNA
sequences . These random genetic changes are likely to be harmful to organisms ; genetic
defects and cancer are believed to be caused by mutations . We have analyzed spontaneous
mutations in the phage M13mp2 lac promoter-lac Za region ; the forward mutations in the
lac promoter-lac Za region that is necessary for a-complementation to restore the Bgalactosidase
activity in E. coli NR9099 were analyzed . Mutant a-complementationdeficient
phages were detected as colorless or pale blue plaques among the wild blue
plaques in the plates containing X-gal and IPTG . Viral single stranded DNA was prepared
from the isolated mutant plaques and sequenced by chain termination procedure of Sanger
et al . using a sequencing kit with a-32P-dCTP . The frequency of spontaneous mutation
was 4 .7 x 10-5 (55/1170000) . Of the 55 DNA samples, sequence changes were detected in
30 samples . The spectrum of these changes comprised transitions, transverslons,
multiple base-deletions, -additions, single base-deletions, and -additions . About a
half of these mutations were single base additions and deletions in several hot spots .
Analysis of the local DNA sequences suggests that the intensity of these hot spots
depends on structural features of the DNA, i .e ., runs of more than four identical bases .
The cause of these spontaneous mutations is, therefore, suggested to be slippage errors
by DNA polymerase of the host bacterium E . coli .
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
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