Environmental and Molecular Mutagenesis - Legacy Tobacco ...

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1989 EMS Abstracts 219
Notes
EVALUATION OF FURAN-INDUCED GENOTOXICITY AND CELL PROLIFERATION IN RAT AND HOUSE HEP-
ATOCYTES . D .H . Wilson, and,. .B .E . Butterworth, CIIT, Research Triangle Park, NC (USA) .
Initial observations from bioassays by the National Toxicology Program indicate
that furan produces hepatocellular carcinomas in male F344 rats and in male and female
B6C3F1 mice . Although furan is negative in the Ames test, the profile of autations
produced in oncogenes from furan-induced tumors are different from those of spontaneous
tumors (Reynolds et al, Science 237, 1309 (1987)) . To investigate the possible
mechanisms by which furan causes tumors, genotoxicity, as indicated by DNA repair, and
chemically-induced cell proliferation were examined . Prijary hepatocyte cultures
from male F344 rats were incubated with faran and 10 LCi/ml H-thymidine . DNA repair
as unscheduled DNA synthesis (UDS) was quantitated autoradiographically as not grains
per nucleus (NG) . Concentrations from 1 YH to 10 aH furan yielded no UDS in vitro . To
examine UDS in vivo, furan was administered by gavage to male rats
(5, 30 and 100 mg/kg) and to male B6C3F1 mice (10 . 50, 100 and 200 ag/kg) twelve hours
prior to isolation of hepatocytes . No increase in NG was observed in hepatocytea from
furan-treated animals compared to r3ontrols . Cell proliferation in vivo was evaluated
by autoradiographic analysis of H-thymidine incorporation in cultures of primary
hepatocytes isolated 36 hours after a single gavage administration of furan (30 mg/kg)
to male rats . Hepatocytes from vehicle treated rats had a labeling index (LI) of
0 .2+0 .1X while that of furan-treated rats was 17+5% . Taken together, available dats
suggest that the distinct profile of oncogenes from furan-induced tumors may have
resulted from enhanced susceptibility of those genes to mutations related to forced
cell proliferation rather than from direct mutagenesis by furan . Puran-induced cell
proliferation may also have been a driving force in tumor promotion and progression .
636
THE CYTOGENETIC EFFECI'S OF INTRODUCING RESTRICTION ENZYMES INTO CHO CELLS
USING ELECTROPORATION. RA Winegar H.W. Chung, J .W. Phillips and W.F. Morgan, Laboratory of
Radiobiology and Environmental Health, University of California, San Francisco, CA (USA)
Studies using restriction enzymes to examine the mechanisms of cytogenetic damage have been hampered
by the inefficiency of available permeabilization techniques . We have used cell electroporation with great
success to permeabilize Chinese hamster ovary (CHO) cells for the introduction of restriction enzyptes . Cells
electroporated in the presence of restriction enzymes show an extremely high frequency (>90%) of aberrant
njetaphases as well as a dramatic decrease in cell survival. Electroporation itself caused no increase in
aberrant chromosomes and had only a slight effect on cell survival . The isoschaomer pair Msp I and Hpa II
was used to determine whether restriction enzymes act with the same sprcificity within a cell as in vitro . Both
enzymes recognize the DNA sequence CCGG, but whereas Hpa II will not cleave this sequence if the internal
cytosine is methylated, Msp I will cleave regardless of the methylation status of the internal cytosine . As
expected, since this sequence is heavily methylated in mammalian cells, Msp I was greatly more effective than
Hpa II at inducing chromosome aberrations . Although restriction enzymes are very effective at inducing
chromosome aberrations, experiments using a large number of different enzymes and several different harvest
times indicated that restriction enzymes do not induce sister chromatid exchanges . This is consistent with
previous reports that agents that produce double-strand breaks do not induce sister chromatid exchanges .
Work supported by the Office of Health and Environmental Research of the U .S. Dept of Energy under
contract DE-AC03-76•SF01012 .
637
ISOLATION OF cONAs ASSOCIATED WITH TUMOR SUPPRESSOR GENE FUNCTION IN SYRIAN HAMSTER
EMBRYO CELLS . R .W . Wiseman, J .C . Montgomery, J . Hosoi, E .W . Hou, J .E . Bisi, and
J .C . Barrett, Laboratory of Molecular Carcinogenesis, National Institute of Environmental
Health Sciences, Research Triangle Park, NC 27709 .
Loss of a tumor suppressor gene function appears to be a critical step in Syrian
hamster embryo (SHE) cell neoplastic transformation in vitro . In order to understand
the function of these genes, we have isolated preneoplastic clonal variants
from 2 imnortal SHE cell lines that have either retained (supB+) or lost (supB-) the
ability to suppress the tumorigenicity of the BP6T tumor line in cell hybrids . cONA
probes prepared from polyA+ RNA of supB+ and supB- cells have been used to screen a
supB+ Lambda Zap cDNA-library for clones that are preferentially expressed in supB+
cells . cONAs for at least 4 independent genes have been isolated, and DNA sequence
analyses revealed that 2 of these encode proal(II) and al(IX) collagens, which are
normally expressed in chondrocytes . Another cDNA was unrelated to any known gene
while the fourth cDNA was homologous to the mouse H19 gene, which is developmentally
regulated in liver and muscle cells . In complementary studies for the identification
of human tumor suppressor genes . Syrian hamster tumor cells have been utilized as
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