Combined Actions and Interactions of Chemicals in Mixtures

DNA, and that the cell was capable of (correctly or incorrectly) repairing the DNA
damage. UDS is most often measured in mammalian cells in culture, usually
primary hepatocytes, but may also be measured in vivo (see below). DNA repair
can also be measured in the comet assay (see below).
7.2.5.2 Assays detecting point mutations
Point mutations in bacteria
The Salmonella/mammalian microsome assay (Ames test) is by far the most widely
used in vitro assay. The Salmonella assay is particularly efficient in detecting
genotoxic carcinogens that produce point mutations, such as aromatic amines, nitro
compounds, polycyclic aromatic hydrocarbons, and a number of other chemicals
able to interact with DNA. Carcinogens that operate through other mechanisms,
such as a number of halogenated compounds or inorganic compounds are not
detected in the assay. The Salmonella assay has been used to measure the
mutagenic activity in samples of numerous complex environmental mixtures (Houk
& Waters 1996) including cigarette smoke condensate, automobile exhaust,
ambient air, industrial wastes, drinking water, polluted soil and urine from people
exposed to potential genotoxins. In addition, this assay has been used to test
extracts from food contact materials, such as plastic (Binderup et al 2002a,
Feigenbaum et al. 2000). In a number of studies bio directed fractionation has been
used to identify single genotoxic compounds in the mixture (Marvin 2000, Dobias
et al 1999, Marvin 1999, Brooks 1998). Advances in the molecular analyses of
mutations in Salmonella have suggested that the mutation spectra produced by
these complex mixtures are reflective of the predominance of a single (or a few)
mutagenic chemical class (DeMarini et al. 1993).
Recently, new genetically modified tester strains with specific changes in genes
coding for metabolic enzymes or repair genes have been developed. Also, strains
with human metabolic enzymes inserted have been constructed. These strains have
enhanced sensitivity for specific chemical classes (e.g. nitro- and amino aromatic
compounds (Josephy et al. 1995), alkylating compounds (Yamada 1997) or
compounds inducing oxidative damages (Josephy et al. 1997).
Point mutations in mammalian cells
Widely used cell lines include L5178Y mouse lymphoma cells, Chinese hamster
ovary (CHO) cells and Chinese hamster V79 cells. Commonly used systems work
by selecting for the loss of function of a gene product (enzyme), which is
inessential for the survival of the cells in culture. Three selective systems have
been widely used in mammalian cell mutagenicity tests:
• Resistance to 6-thioguanine (6TG) and 8-azaguanine resulting from lack of
hypoxanthine phosphoribosyl transferase (HPRT) enzyme activity.
• Resistance to trifluorothymidine (TFT) and 5-bromodeoxyuridine (5BrdUrd)
resulting from lack of thymidine kinase (TK) activity.
• Resistance to ouabaine (OUA) resulting from lack of Na + /K + ATPase enzyme
activity.
7.2.5.3 Cytogenetic assays
Chemicals that induce structural chromosome aberrations are termed “clastogens”,
and the test systems used for their detection are called cytogenetic assays.
Cytogenetic tests are based upon detection of certain chromosome changes
observed in the light microscope. The most commonly used standard cytogenetic
assays are chromosomal aberration analyses (CA), sister chromatid exchanges
(SCE), and the micronucleus assay (MN).
86