Environmental and Molecular Mutagenesis - Legacy Tobacco ...

18 1989 EMS Abstracts
Notes
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
DNA ADDUCT FORMATION IN RELATION TO TUMORIOENESIS IN MICE
CONTINUOUSLY ADMINISTERED 2-ACETYLAMINOFLUORENE OR 4-AMINO-
RIPHENYL Frederick A . Beland, Nancy F . Fuperton, M . Matilde Marques. William B .
Melchior, Jr., Beverly A . Smith and eMiriam C . Poirier National Center for
Toxicological Research, Jefferson, Arkansas 72079 and •National Cancer Institute,
Bethesda, Maryland 20892
Continuous administration of 2-acetylaminofluorene 12-AAFI or 4-aminobiphenyl (4•
ABP/ to female BALB/c mice results in the formation of liver and bladder tumors . We
have compared the relationship between the concentration of DNA adducts in these target
tissues following one month of treatment and the reported tumor incidences after two
years of dosing . One adduct, N-ldeoxyguanosin-8 y1F2•aminofluorene (dO-C8-AF1, was detected
after 2-AAF feeding, and at each of the seven doses . the adduct concentration in
the bladder was two- to four-fold higher than in the liver . Two major adducts, one of which
was N-ldeoxyguanoain-8 y11-4-aminobiphenyl ldO-CB-ABPI, were found in mice treated with
4-ABP, and In contrast to 2-AAF, the adduct levels were five- to eight-fold higher In the
liver than in the bladder. At doses producing equivalent liver tumor incidences, hepatic
4-ABP adduct levels were higher than those induced by 2-MF, which suggests that
2-AAF adducts are more efficient at inducing tumors than 4-ABP adducts . In order to
investigate differential processing of these adduct. . pBR322 DNA, modified with dO-C8-
A13P and d0-C8-AF . was transformed Into 8OS-repair induced F. coH. Although both
adducts induced a similar pattern of transversion and transition mutations, d0-C8-AF also
caused deletions . Thus, thee turoorigenic and mutagenic potential of aromatic amine adducts
appear to be compound specific .
USE OF THE POLYMERASE CHAIN REACTION TO AMPLIFY A SEGMENT OF THE ~$ GENE OF
Salmonella FOR DNA SEQUENCE ANALYSIS . Douglas A . gelll, Jaaes E . Lae2, tvnhi and David M
. DeMarini . 1National Research Council, U .S . EPA, RTP, NC 27711 ; 2Duke
University Medical Center, Durham, NC 27710 ; and 3U .S . EPA, RTP, NC 27711 (U .S .A .) .
Previous DNA sequence analyses of revertants of the hisD303l gene of $ . t,yphimurium
TA98 or TA1538 have employed either a colony hybridisation technique (Cebula et al . .
Environ . Mol . Mutagen . 11, Suppl . 11 :21 ; 1988) or traditional cloning procedures
(Fuscoe et al ., Mutat . Res . 201 :241 ; 1988) . pa have explored the polymerase chain
reaction (PCR) to see if it might permit more rapid processing of revertants for DNA
sequence analysis than do the other methods . Briefly, a revertant is streaked onto
minimal medium supplemented with biotin . After grovth, a colony is boiled for 5 min
in 200 yl of TE buffer and then centrifuged for 5 min . The supernatant contains the
Salmonella genomic DNA used for the PCR . The two amplification primers (AP) flank a
327-bp segment that contains the hi,gD3Q52 mutation approximately in the center . The
sequence of AP1 is : 5' GTC TGA ACT ACT GGT CAT CG 3' ; AP2 is : CCC TOG TM TCG CAT
CCA CC . The reaction is performed essentially as described for Taq polymerase in the
Perkin Elmer Cetua GeneAmp KitTM using 10 µl of Salmonella genomic DNA/200-pl reaction
volume . Amplification of ds-DNA is obtained using a 1 :1 ratio of the primers (50
pmoles of each) and 30 cycles of an automatic thermocycler ; amplification of ss-DNA is
obtained using a 1 :100 (0 .5 :50 pmoles) ratio of primers and 36 cycles . The amplified
DNA is purified by ultrafiltration in dH20 in an Amicon CentriconTM 30 microconcentrator,
followed by resn• .p,na3on in 9 pl of sequencing buffer . The feasibility
of employing nested primers for sequencing the amplified DNA is being evaluated .
(Abstract of proposed presentation that does not necessaril'y reflect U .S . EPA policy,)
VIDEO MICROSCOPY AND DIGITAL IMAGE PROCESSING OF PRENATAL BRAIN CELLS EZPOSED
TO LEAD NITRATE . Maxwell A . Bampong, Biology Department, Norfolk, VA 23504 .
The effect of glutamic acid on lead-induced morphological changes io cultured
brain cells derived from prenatal mouse, was examined . Fluorescence microscopy,
video-enhanced/intensified microscopy and digital image processing were used to
monitor dose-dependent, qualitative and quantitative changes in cells exposed to
lead nitrate and incubated in the presence or absence of 102 glutamic acid . Leadtreated
cells without concurrent glutamic acid exposure were characterized by
cell rounding, spreading and extensive cytoplasmic vacuolation . Thirty ∎inute
exposure to S ug/ml of rhodamine 123, resulted in fluoreicently stained cytoplasm
as well as nuclei . Video-intensified microscopy confirmed lead-induced
degenerated nuclear membrane . Other morphological changes observed to be associated
with lead exposure included dendrite retraction, globular shaped cells
with the attending increased shape factor (a fraction for estimating the amouni
by which a cell varies from a circle), and increased total cell area . Theai
stereological changes in lead-treated calls, were dose-dependent . Cells incubated
concurrently in lead and glutamic acid medium, regardless of lead concentration,
were mostly spindle in shape, bi- or multipolar, with very low shape factor
values . These data from fluorescence and video-intensified microscopy suggest
that intracellular membranes were the primary targets of lead action . Intracellular
digestion leading to extensive vacuolation was effected by lead impact on
lyaosome, surface membrane disruption was responsible for changes in cell shape
and the appearance of rhodamine 123 in nuclear region resulted from lead induced
nuclear membrane degeneration .
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