Environmental and Molecular Mutagenesis - Legacy Tobacco ...

50 1989 EMS Abstracts
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
Notes radiation or benzo[a]pyrene diolepoxide and then allowed to replicate in CV-1 monkey
cells, mutations are induced in the mutagenesis target gene (the bacterial auoF gene)
of the vector . DNA sequence analysis of these mutations reveals that the two
mutagens induce very different spectra of mutations . By comparing the mutational
spectra with the spectra of damage induced by the two agents, we have been able to
deduce cellular mutational mechanisms . Recently, we constructed a series of
polyomavirus-based vectors that replicate in rodent cells to facilitate the analysis
of the role that cellular DNA repair processes play in mutational specificity . With
these vectors we have begun to analyse the specificity of UV mutagenesis in repairdeficient
mouse cells . We have also studied mutagenesis in an in vitro DNA
replication system . The p2189 vector can be completely replicated in this cell-free
system in the presence of SV40 T-antigen . Undamaged vector DNA is replicated with
high fidelity in this system . UV-damaged vector DNA is also replicated, but less
efficiently . It appears that mutagenesis occurs as a consequence of replication of
the UV-damaged templates in vitro . Preliminary results suggest that the spectra of
mutations induced by UV in vitro resemble those induced in vivo . Thus, the in vitro
system may prove to be particularly useful in the molecular analysis of mutation
induction in mammalian cells .
139
STABLE EXPRESSION OF P450IIB1 AND P450IA1 CDNA IN V79 CHINESE HAMSTER
CELLS APPLIED TO MUTAGENICITY TESTING
J . Doehmer, S . Satish, H .R .Glatt, and F . Oesch
Institut fUr Toxikologie, Johannes Gutenberg-Universit8t,
D-6500 Mainz, FRG
cDNA encoding P450IA1 and P450II81 were recombined with the SV 40
eukaryotic vector . The recombinant plasmids were transferred into V79
Chinese hamster cells by the calcium/phosphate co-precipitation
procedure using the neomycin phosphotransferase gene as selective
marker . Several cell clones were obtained . Cell clones were
characterized by Southern-, Northern-, and Western-blotting . The
enzymatic acttivity was determined using P450 specific substrates like
7-pentoxyresorufin in the case of P450IIB1 and 7-ethoxycoumarin in the
case of P4501A1 . Specific activities were found to be in the same range
typical for uninduced liver . Cytotoxicity and mutagenicity studies
revealed that P450 producing cells respond to the exposure of
cyclophosphamide, benzo(a]pyrene, and trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene
in a dose dependent manner . The mutation rate
also correlated with the different levels of activity contained in the
various cell lines . So far, the enzymatic activity remained stable in
these cell lines for more than one year .
The project is supported by the Deutsche Forschungsgemeinschaft (SFB
302 "Kontrollfaktoren der Tumorentstehung") .
140
EFFECT OF SIMULATED ACTIVATED SLUDGE TREATMENT USING BENCH-SCALE BIOREACTORS ON THE
MUTAGENICITY OF MUNICIPAL WASTEWATER . J . U . Doerger, J . R . Meier, R . A . Dobbs and G .
N . Stelma, U . S . Environmental Protection Agency, Cincinnati, Ohio, 45268 .
Previous studies of mutagens in municipal wastewater have shown that following
activated sludge treatment the level of extractable organic matter had decreased .
However, the level of mutagenic activity had not decreased substantially with treatment .
Two possible explanations for this finding were postulated : the bioreaction process
increased the number or potency of the mutagens, or the process removed non-mutagenic
compounds more efficiently than it removed autagens . In order to have a controlled
system for comparing pre- and post-treatment samples, bench-scale bioreaction studies
were performed . Primary effluent was inoculated with aerobic organisms from the aeration
basin and incubated at 23°C with aeration for 24 hrs . The extracts were evaporated
and redissolved in DMSO for subsequent autagenicity testing using the Ames Salmonella
assay with tester strain TA98 with and without S9 activation . The results of the
bench-scale bioreactor were consistent with data derived from treatment plant studies .
Total mutagenic activity was not significantly changed with incubation ; however, the
specific mutagenic activity (his+ revertants/mg) of post-incubation extract did increase
approximately 50% . The weight of the extracts decreased 35% . Viable cell counts of
the inoculated wastewater indicated 40-fold increase after the incubation . These data
suggest that the compounds responsible for mutagenicity in a municipal wastewater are
less biodegradable by activated sludge treatment than the bulk of the extracted compounds
. Consequently, improved biological and/or chemical-physical processes may be
required to remove the mutagens . (This abstract does not necessarily reflect EPA
policy) .
50869 3562