Toxicology of Industrial Compounds

Alternatively, the alkylated N-terminal valine of haemoglobin can
selectively be cleaved off by a modified Edman degradation with
pentafluorophenyl isothiocyanate (PFPITC). Since alkylation of the amino
group favours the reaction, conditions can be selected to exclusively
liberate alkylated N-terminal amino acids whilst leaving the non-adducted
N-terminal valine intact (Törnqvist et al., 1986). The resulting
pentafluorophenyl thiohydantoine (PFPTH) derivative can be extracted and
quantified by GC/MS (Figure 6.3).
Immunological methods
Immunological methods have been developed for the quantification of
some adducts of aromatic amines, polycyclic aromatic hydrocarbons and
alkylating agents. However, these methods involve a couple of time
consuming steps for the isolation of an appropriate antibody. The
respective haemoglobin adduct has to be chemically synthesised, an animal
has to be immunised with the modified haemoglobin and, later on,
polyclonal antibodies can be isolated from the blood of the immunised
animal. In order to produce monoclonal antibodies, which normally have a
better specificity and sensitivity, spleen cells of the immunised animal are
fused with myeloma cells and the antibodies can be isolated from the cell
culture.
The methods for the determination of adducts include competitive
radioimmunoassays and solid phase assays (ELISA, USERIA). The protein
is partially hydrolysed, adsorbed on a solid surface and treated with the
primary antibody. An anti-antibody which is directed against the primary
antibody, radiolabelled, or conjugated to a fluorescent dye or an indicator
enzyme, is added and the amount of bound label is quantified (Santella et al.,
1986; Lee and Santella, 1988).
DNA adducts
Physical methods
Aromatic amines and nitroarenes
P.SAGELSDORFF 77
The hydroxylamines produced by enzymatic hydroxylation of aromatic
amines or by reduction of nitrosoarenes are further conjugated (Osulphatation,
O-acetylation, O-glucuronidation). The conjugates can
decompose to the respective nitrenium ions which add predominantly to
the C8 of guanine.
Similarly to protein adducts of these compounds, the adducts can be
liberated from DNA by alkaline hydrolysis or hydrazinolysis, extracted and