Environmental and Molecular Mutagenesis - Legacy Tobacco ...

142 1989 EMS Abstracts _ 410
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
NOteS- "" y'"RECOMMENDED 1TAMM PROTOCOLS BASED ON A SURVEY OF CURRENT PRACTICES IN GENOTOXICITY
TESTING IABOR{RORIES . E .R . Nestmann(1), S .H .H . Swierenga, R .L . Brillinger(1), and
J .P .W. Gilman ~.Directorate, Health Protection Branch, Ottawa, and (1) CanTox
Inc ., 627 Lyons lane, Suite 200, Oakville, (Canada)
The most commonly used genotoxicity assays for cultured mammalian cells are
mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell
transformation . Since their inception, protocols for these assays have been modified
in various laboratories . It has been observed that minor but potentially significant
method modifications frequently remain unpublished (Swierenga et al ., J . Tiss . Cult .
Meth . 8 :7, 1983) but should be considered in the development of standard protocols .
The present study was undertaken to determine the current "state of the art" for these
tests . Detailed questionnaires on culture conditions and testing protocols for both
stock and test cell populations were designed with the assistance of an international
advisory committee and sent to all research and contract laboratories that could be
identified in Canada, USA, and Europe . Responses from 425 completed questionnaires
were analyzed to determine the most commonly used approach and modifications for each
procedural step . As expected, the results show a large degree of interlaboratory
variation . Detailed protocols for conducting each assay have been prepared and
include : stepwise instructions, precautionary measures and practical solutions to
common problems associated with each assay ; recipes for media and solutions ; formulas
for quantifying genotoxic responses ; reference lists of related assays ; guidelines for
interpretation ; and discussions of the applications, advantages and disadvantages of
each test .
411
CRITERIA FOR ASSESSMENT OF GENOTOXICITY DATA WITH RESPECT TO IN VIVO MUTAGENICITY AND
MECHANISM OF CARCINOGENICITY . E .R . Nestmann and L .D . Kier, CanTox Inc ., Oakville
(Canada), and Monsanto Co ., St . Louis (U .S .A .)
Evaluation of genotoxicity databases often is complicated by a mixture of positive
and negative results . We have identified general criteria that can be used in a
"weight of evidence" assessment . Evidence for Jja y1yQ genotoxicity is strenothened by
any or all of the following : induction of genetically stable effects (i .e ., mutation)
rather than indicator endpoints of DNA damage (e .g ., SCE, UDS) ; activity for multiple
endpoints ; in vivo (rather than only in vitro) effects ; induction by appropriate route
of exposure and at not overtly toxic doses ; structure and pattern of activity related
to known mammalian mutagens ; strong responses . The case for j,p yjys genotoxicity is
weakened by : negative in vivo results ; positive results only In vitro, especially
activity that is reduced or eliminated in the presence of metabolic fractions ; activity
that is only observed under conditions known to cause artifacts (e .g ., high
cytotoxicity or osmolality, low pH) . Evidence for carcinogenesis through a genotoxic
mechanism is strengthened by : consideration of the above criteria for in vivo
genotoxicity ; correlation of in vivo genotoxicity and carcinogenicity findings (e .g,
tissue or species specificity ; route) ; similar pattern of response as chemically
related carcinogens . Evidence for a nongenotoxic mechanism is strengthened by :
activity only in tests with low specificity ; other evidence for nongenotoxic or
promoter mechanism of carcinogenesis . (Supported in part by the AIHC, CMA and
ILSI/RSI ; contributions of the AIHC Mutagenicity Subcommittee are gratefully
acknowledged .)
412
MOLECULAR ANALYSIS OF Zl= MUTATIONS ARISING jg VIVO IN HUMAN T-LYMPHOCYTES, J .A . Nicklas,
T .C . Hunter, J .P . O'Neill, L . Recio, D . Simpson, T .R . Skopek, and R .J . Albertini, VRCC
Genetics Lab, Univ . of Vermont, Burlington, VT and CIIT, Research Triangle Park, NC .
hort and T-cell receptor (TCR) Southern blot analyses were performed on 96 wild type
and 330 somatic h= mutant clones from three normal individuals . h= analysis showed
that 16 .0, 16 .5 and 9 .6% of the mutations had visible b= structural alterations in the
3 individuals, respectively . The breakpoints of the deletion mutations were spread across
the gene in proportion to length (r- .94) with 0 .73 breaks/kb, allowing an estimate of the
nearest flanking vital genes of 18kb 5' and 26 .5kb 3' . TCR analysis determined that 91,
85 and 85% of the mutants were i .ndependent T celi clones (i .e . had different TCR gene
rearrangements with TCRj8 or y gane probes) . Doublets, triplets, quadruplets and one
nonamer ∎et of sibling clones were observed . The average persistence of a clone was
2 .8,2 .9 and 1 month, respectively although the nonamer siblings persist after 2h years .
We have previously described the extensive proliferation of a T call clone in another
normal individual resulting in an elevated mutant frequency with greater than 90% sibling
clones . One individual had 6 mutant clones with the saue apparent exon 2-3 deletion and
new size axon 1 fragment ; by TCR analysis 4 are sibling clones while 2 share only TCRB
gene rearrangements . This latter fact suggests an intrathymic mutation or extra-thymic
TCRy gene rearrangement . This same apparent deletion was found in a mutant from another
individual suggesting a"hotspot" for mutation . We found two cases of an apparent TCR
clone (i .e . the same TCR gene rearrangements) containing 2 different b= mutations which
is evidence for sensitivity of a dividing TCR clone to mutation . We are currently
sequencing the mutants which did not show hp= gene alterations to define a spectrum of
spontaneous" somatic mutation in man . Supported by NCI CA30688 .
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