Environmental and Molecular Mutagenesis - Legacy Tobacco ...

146 1989 EMS Abstracts .
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
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Notes - NOLECUIJ1It~YSIS Y OF N-HYDROXY-2-ACETYLMINOFUlONF1V~-INUCED MVfATIGNS IN
CHIN~SE CELLS LnCRING DiA ERCISION REPAIR . R .T. Okinaka, S .L . Ansick,
and G .F . StrQis~i..Genetics Group, Life Sciences Division, Los Alamos National
Laboratory, LZSs-amos, M 87545 (USA) .
It is generally believed that damage to DNA can be fixed as heritable mutations
that are a conssquence of errors in repair or replication through the
damaged sites . In order to better understand the role of DNA replication in the
fixation of mutation, we have employed in our studies a DNA excision repairdeficient
Chinese hamster cell line (CH0 W-5) . initial experiments were
designed to define the magnitude of possible deletion mutations induced at the
X-linked hypoxanthine-guanosine phosphoribosyl transferese (HPRT) locus
N-hydroxy-2-acetylaminofluorene (N-OH-AlLLr) using restriction enzyme digesti~
Southern blotting and probing techniques (with a cloned hamster V79 cell HPRT
cDNiA) . However, our observations indicate that none of the N-OH-AAF-induced
UV-5 mutants analyzed to date contain large deletions or rearrangements at this
genetic locus . In order to define the molecular nature of these mutations, we
have recently employed a modification of the polymerase chain reaction (PCR)
technique in conjunction with direct sequencing strategies . Preliminary results
indicate that some of these mutants contain truncated HPRT s*As, possibly the
consequence of small deletion events or processing errors in introNexon
splicing . (This research is supported by the U .S . Department of Ehergy under
contract W-7405-ENC--36)
422
423
OVEP.VIEW OF IN VIVO MATQIALIAN TESTING SYSTEMS . F . B . Oleson, Bristol-Myers Company,
Syracuse, New Yorc-(USA) .
Both somatic and germ celle can be targets for genetic damage . In vivo mammalian
assays for both types of damage will be described . Germ cell assays incfude spermatocyte
cytogenetics or the new biochemical specific locus assay (currently under
validation), specific locus and heritable translocation assays, and the rodent
dominant lethal assay . Somatic cell assays have historically focused on the bone
marrow, including cytogenetic studies for chromosomal aberration (CA) or, more
recently, the erythrocyte micronucleus test . Other in vivo tissue-specific genotoxicity
assays will briefly discussed, including 1) sistar c4rcomatid exchange (SCE)
in bone marrow, 2) in vivo/in vitro hepatocyte DNA repair, 3) host mediated and 4)
rodent lymphocyte CATSCE tests . Promising new test systems will also be presented
such as the use of tranegenic mice for studies of oneogene activation and the single
cell gel (SCG) DNA fragment electrophoresis assay . International regulatory
guidelines for drug and chemical safety evaluation uniformly recommend one in vivo
study be performed as part of a battery of mutagenicity tests . Bone marrow CK-or
micronucleus assays are the recommended systems . Recent validation studies support
the micronucleus test as the primary in vivo screen . The sensitivity and efficiency
of these systems have been improved Sy moaffication of dosing/sampling time design,
use of automated scoring techniques and the use of mouse peripheral blood for
micronucleus testing . Finally, the rationale and scientific justification for the
integration of multiple genetic toxicology endpoints into standard subacute (7- or
30-day) rodent toxicology studies will be outlined .
424
AUTOMATION OF MOUSE PERIPHERAL BLOOD MICRONUCLEUS SCORING . F .B . Oleson . S .M . Getman,
H . Mahran and G . Jenkinson, Bristol-Myers Company, Syracuse, NY (USA), and Cambridge
Instruments, Inc ., Deerfield, IL (USA) and Cambridge (UK) .
Procedures for the automated scoring of fluorescent stained mouse peripheral blood
smears have been developed . . Peripheral blood smears stained with acridine orange are
analyzed under high resolution microscopy in combination with a Cambridge Instrumants
Quantimet 520 Image Analyzer . Orange-fluorescing polychromatic erythrocytes (PCEs)
and yellow-fluorescing micronuclei are enumerated via the image analyzer . The total
erythrocytes scanned in all fields analyzed is automatically estimated using a
standard per field cell count . A motorized stage and autofocus allow for the rapid .
analysis of individual fields for the total number of PCEs and micronucleated PCEs
(MN-PCEs) . For each slide, the percent !Qi-PCEs and percent PCEs in total erythrocytes
scanned are calculated and automatically tabulated by computer . Validation
data comparing manual and automated micronucleus scoring will be presented including
estimates of automated counting errors in the MN-PCE and PCE frequencies . Counting
speed, limitations and problems with automated procedures will be discussed . Manual
peripheral blood micronuclaus scoring for a standard study using 30 animals (1000
PCEs/animal) and one evaluation time can be reduced from appror.imately 10 days to 2-3
days using these automated procedures with no significant difference in results .
These procedures should provide for improved assay sensitivity and reliability since
appreciably more cells can be scored per animal than could be reasonably accomplished
via manual scoring techniques .
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