Toxicology of Industrial Compounds

78 METHODS FOR THE DETERMINATION OF REACTIVE COMPOUNDS
quantified by HPLC with fluorescence or electrochemical detection. In
order to improve the sensitivity the extracted adducts can be derivatised
with electrophores and analysed by GC/MS (Bakthavachalam et al., 1991;
Lin et al., 1991).
Polycyclic aromatic hydrocarbons
The diol epoxides enzymatically produced from polycyclic aromatic
hydrocarbons mainly adduct at the exocyclic amino group of guanine. The
adducts can be liberated from DNA by acid hydrolysis, extracted and
quantified by HPLC with fluorescence or electrochemical detection or by
GC with electron capture detection or GC/MS after suitable derivatisation
with electrophores (Rahn et al., 1982; Shugart and Kao, 1985; Weston et
al., 1989).
Alkylating agents
Alkylating agents mainly alkylate the N7 of guanine but also give rise to
the formation of other N- and O-alkyl nucleobase adducts. The DNA bases
are liberated by hydrolysis and analysed for the presence of adducts by
HPLC with electrochemical detection or they are extracted, derivatised
with electrophores and analysed by GC/MS (Minnetian et al., 1987; Groot
et al., 1994). Some alkylating agents and small epoxides lead to the
formation of cyclic nucleobase adducts which exhibit strong fluorescence.
Enzymatic or acid hydrolysis can be used for the liberation of the DNA
constituents and the fluorescent adducts can be analysed by HPLC with
fluorescence detection (Fedtke et al., 1990; Steiner et al., 1992a).
Immunological methods
Immunological methods for the determination of DNA adducts essentially
follow the procedure as outlined already for protein adducts: generation of
an antibody, absorption of the DNA on a solid surface, incubation with the
antibody and a labelled anti-antibody. However, for the production of the
antibody an additional step has to be performed. The immune system
normally does not respond to small molecules. Therefore, the chemically
synthesised base or nucleoside adduct has to be coupled to a carrier protein,
in order to obtain an immunogen (Perera et al., 1986; Santella, 1988;
Poirier, 1993).
Postlabelling
One of the most popular assays for determination of DNA adducts is the
postlabelling assay. The DNA is enzymatically hydrolysed to the four