Combined Actions and Interactions of Chemicals in Mixtures

Structural chromosomal aberrations (CA)
Two types of cells are routinely used, Chinese hamster ovary (CHO) cells and
human lymphocytes, which are stimulated to divide in vitro. The assay is
conducted in the absence and presence of exogenous metabolic activation. To
visualise the chromosomes, the cells are arrested in metaphase by adding a spindle
inhibitor (e.g. colchicine), stained and examined in the microscope. Large, visible
aberrations are recorded. They can be of the “chromatide type” involving one
chromatid or “chromosome type“ involving both chromatids. Discontinuity in
stained regions of chromosomes can be classified as “gaps” or “breaks”. Deleted
material may appear as fragments in metaphase preparations. Chromosomal
breakage is necessary for chromosomal rearrangements. A chromosomal exchange
results, when the broken ends of the same or different chromosomes rejoin in an
aberrant manner. An inversion results from two breaks in the same chromosome
with the broken pieces U-turned before rejoining. A translocation results from an
interchange between non-homologous chromosomes. As mentioned above
translocations are considered to be some of the most biological significant
aberrations, but it is almost impossible to score using current cytogenetic standard
techniques. CA can also be measured in vivo in different animal species, and have
been used in several biomonitorings studies (Sorsa et al. 1994, Knudsen et al.
1999, Bogadi-Sare et al. 1997, Brenner 1996, Tompa et al. 1994, Sroczynski et al.
1994).
Fluorescence in situ hybridisation (FISH)
Fluorescence techniques such as FISH or “chromosome painting” (Pinkel et al.
1988) may significantly improve the methods currently used for studying chronic
exposure to low levels of clastogenic agents in complex mixtures. Fluorescent
probes have been developed that can identify specific chromosomes or part of
chromosomes through in situ hybridisation. This technique makes it possible to
determine structural chromosomal aberrations like transversions, which, as
mentioned above, is almost impossible to measure by conventional cytogenetic
assays. Also, aneuploidi, for which no guideline tests exist, can be detected by this
method. The limitations of FISH are that only a few chromosomes at a time can be
labelled because of restricted ability to differentiate multiple fluorescent signals,
and that the availability to specific karyotypes is limited.
Micronucleus assay (MN)
The micronucleus can be performed in vitro, and international guidelines will
presumably be available in the near future. The test is described below, as an in
vivo assay.
Sister chromatid exchange (SCE)
The induction of DNA lesions by genotoxic agents leads to the formation of sister
chromatid exchanges (SCE), which may be related to recombinational or
postreplicational repair of DNA damage. SCE represents the interchange of DNA
replication products at apparently homologous loci. The exchange process
presumably involves DNA breakage and reunion, although little is known of its
molecular basis. SCE are revealed by a “harlequin pattern” of differential stained
chromatid segments in chromosomes from cells grown in the presence of BrdUrd
(bromodeoxyuridine) for two rounds of DNA replication. After treatment with a
spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase like stage (cmetaphase)
cells are harvested and chromosome preparations made. After staining
with fluorescence-plus-Giemsa (FPG) technique, so that one chromatid is more
lightly stained than the other is, SCE can be observed by conventional light
microscopy. SCE can also be measured in vivo in a variety of animal species, and
have been used as a biomarker in human populations exposed to complex mixtures
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