Environmental and Molecular Mutagenesis - Legacy Tobacco ...

413 ~ 1989 EMS Abstracts 143
DNA SEQUENCE CHARACTERIZATION OF ALKYLATION-INDUCED YERMILION Y{TPANTg IN DROSOPBILA. Notes
t( .J .11 . Nivard, A .PastinkYand E .1f .Voge1, University of Leiden, Leiden (The Netherlands) .
The aim of this study hsi been the characterization of base sequenoe changes induced
by ethylnitrosourea (ENU), ethylmethanesulfonate (Et(8), and methylaetbanesulfonate
(MMS) at the vezm{Zion locus of Drosophila, after treatment of postmeiotie male germ
cells . The analysis included consideration of the position of the carcinogen on the
potency scale for carcinogenicity (TU50 in relation to initial alkylation pattern ;
collaboration with Drs .B .Bartsch and A .Barbin, IARC, Lyon), and alteration of the relative
distribution of primary DNA lesions . Sequence analysis of mutants was performed
using the recombinational screening method by 8eed in combination with dideoxy-sequencing,
and the PCR method . All vermtZion mutatlons induced by ENU and EtE were due to
base-pair changes, and all 22 mutations induced by EIIS represented OC : AT transitions .
With ENU, 29 nucleotide substitutions were found in 26 mutants (3 mutants had 2 basepair
changes) . Of these mutations, 23/29 (79%) were transitions and 6/29 (21%) transversions
. In 18/29 (62%) of the mutations tiC : AT transitions were observed . Our results
support the view that 08-ethylguanine is the premutagenic lesion in DrOsophila after
treatment with EYS and ENU . In the latter case, other products of O-ethylation (02EtT,
04EtT) seem to be involved . Characterization by DNA sequencing of 40 mutations induced
by MMS revealed a decidedly different spectrum in relation to EMS and ENU : 21/40
mutations (53%) were tranversions, 8/40 (20%) deletions and 10/40 (25%) transitions .
Misrepair and indirect miscoding seem the predominant mechanisms of action of high S
value type carcinogens . It is this type of genotoxin for which strongest hypermutabllity
effects are obtained in an excision repair defective background .
414
NA REPAIR TESTS ON FOOD ADT)ITIVBS
N'ichiko Non :,ka
iaboratory of Food Chemistry, Faculty of Agriculture,
Kyushu University, ?iunzoka, Japan .
The liquid Bacillus subtilis rec-assays were carried out 09 25 food
additives, all of which are eurrently used in Japan . Briefl .y, the overniBht
culture of . subtilis strains, H17 eLnd ;d45, was mixed with test
solutions and incubated for 30min at 37'C . After treatment, viable
cells aere co•.i .Vltec2 and the ratio of 5M, .• stu'vi,va.l, coace:ltr,itions were
calculated . 18 additives ((acetone, L-asoorbic acid, bettsoio acid, ethyl
acetate, Food Red No .2(tanaranth), Food 3ed No .3(erythrosine), Food Red
No .106(acid red), Food Yello•,v :To .h(tartrazine), glycerin, hexane, pota,ssium
sorbate, propylene rlycol, nropyl gel3e.te, sacchexin sodium,
socitvn benzonte, sodium dehydro3cetate, sodium nitrate, sodium sl-te .rtrste)
ehovaed D :•', damaging potential e.lthough they had been negative in
the ~ .~nes test . Seven additives (erythorbic acid, Food Green No .3(Fast
Green FCP), hydrogen peroxide, potassium bromate, soflium nitrite, cacao
;i&nent, cochineal) showed D'Ta damaging potential, which had been positive
in the Ames test . These s+tr~-eRi ; ''s,+." ~; the ltj,tt' Bs subtilis
rec-=- se : y 1-~ y be more sensitive than the Ames test . -
415
THE MICRONUCLEUS ASSAY IN LYMPHOCYTES.
H . Norppa, M . Hayashi, and J . Miki-Paakkanen, Institute of Occupational Health, Topeliuksenkatu 41 aA,
SF-00250 Helsinki, Finland .
Cytogenetic monitoring of human populations exposed to genotoxic agents has almost exclusively
relied on the use of peripheral lymphocytas . The sensitivity of the lymphocyte culture system In detecting
in vivo exposure may not always be as good as desired, but it could be improved by more extensive
analyses, method development and automation . In the human lymphocyte micronucleus assay, a major
break-through has been the use of cytochalasin B, introduced by Fenecb and Morley, to idenNfy cells
that have divided once in the culture . The recognition of these eells by the presence of two nuclei has
abolished the earlier problems experienced with differences in cell growth and haa made the scoring of
micronuclei in lymphocytes more accurate . Our experience on human monitoring studies, however, suggests
that - in order to catch the cells after their first division - cytochalasin B treatment has to begin
considerably earlier than originally suggested . As cytochalasin B appears to have some toxic effects on
the lymphocytes, it would be good to have an alternative way to recognize the relevant eell population .
At our laboratory, we have been developing a technique based on S-bromodeoxyuridine (BrdU)
incorporation combined with a monoclonal antibody against BrdU-DNA, in order to produce a method that
could be used as a basis for automated scoring of micronuclei by image analysis . BrdU can also be used
without the antibody by applying a differential staining technique, but the antibody method is more
specific and more suitable for automation, because only labelled nuclei are visible. For both of these
approaches, the timing of BrdU treatment and cell harvest are ths critical points . It Is probable that
micronucleus analysis in lymphocytes, with its rapid scoring and suitability for automation, will be an
important alternative for the traditional analysis of metaphase chromosome aberrations in monitoring
human exposure to genotoxins as well as in screening of elastogens in vitro .
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
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