Combined Actions and Interactions of Chemicals in Mixtures

(Sorsa et al. 1994, Myslak & Kosmider 1997, Karahalil et al. 1998, Gonsebatt, et
al. 1995, Bogadi-Sare et al. 1997, Fucic et al. 1998).
Mitotic recombination in Saccharomyces cerevisiae
In eukaryotic cells, genetic exchange between homologous chromosomes is
generally confined to meiosis. In yeast, recombinational events may also be
detected during mitosis, although the spontaneous frequency is generally at least
1000 times less than the meiotic rate. Mitotic recombination can be detected in
yeast both between genes and within genes. The former is called mitotic crossingover
and gives reciprocal products whereas the latter, called gene conversion, is
mostly a non-reciprocal event. Crossing-over is generally assayed by the
production of recessive homozygous colonies or sectors produced in a
heterozygous strain, whereas gene conversion is assayed by the production of
prototrophic revertants produced in an auxothrophic heteroallelic strain carrying
two different defective alleles of the same gene. Mitotic crossing-over producing
red and pink homozygous sectors can be assayed in D5 and D7, both strains being
heteroallelic for complementing alleles of ade 2. D7 also detects reverse mutations
at ilv 1-92. The most commonly used strains for the detection of gene conversion
are D7 (heteroallelic at trp 5) and JD1 (heteroalleic at his 4 and trp 5). Mitotic
recombination may also be measured in other fungi such as Aspergillus nidulans.
7.2.6 In vivo assays
Short-term in vivo tests have also been used to evaluate the genotoxicity of
complex mixtures (DeMarini et al. 1989, Lewtas et al. 1993, Gallagher et al. 1993,
Gallagher et al. 1990). In these types of tests an organic extract of the mixture - or
sometimes the crude mixture in itself - is administrated to the whole animal, and,
after a few days, tissues or body fluids are collected and evaluated. Indicators of
genetic damage include formation of DNA adducts, DNA strand breaks, DNA
repair, chromosomal aberration, SCE and detection of micronuclei (structural and
numeric chromosomal aberrations). However, only a few in vivo genotoxicity tests
are validated and currently in use:
Tests which measure structural chromosomal aberrations in somatic cells: CA and
MN in bone marrow or MN in peripheral blood, and tests measuring DNA damage
and repair: UDS in the liver. CA and UDS have been described as in vitro tests;
only the micronucleus test will be mentioned here.
7.2.6.1 Micronucleus assay
Micronuclei originate from chromosomal material that has lagged in anaphase and
have not travelled to the appropriate pole of the spindle to be included in the main
nucleus. The fragments are included in a separate nuclear membrane at telophase,
and such a structure is called a micronucleus. Micronuclei consist mainly of
acentric fragments, but may also consist of entire lagging chromosomes as a result
of spindle disturbances. Fluorescent probes for kinetochores have been developed
(Degrassi & Tanzarella 1988) and this allows discrimination between micronuclei
with chromosomal fragments and micronuclei with whole chromosomes.
Therefore, the micronucleus test can also detect some aneugenes. Micronuclei can
occur in any cell type of proliferating tissue. They are, however, most easily
recognised in cells lacking the main nucleus, namely erythrocytes. Micronuclei can
be scored using flow cytometry, and this technique significantly improves the
sensitivity of the method.
7.2.6.2 COMET-assay
The application of the COMET-assay or single cell gel electrophoresis assay (SCG
assay) to measure the genotoxic effect of complex environmental mixtures is
increasing, and it might be a promising test for future evaluations. Singh et al.
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