Toxicology of Industrial Compounds

84 METHODS FOR THE DETERMINATION OF REACTIVE COMPOUNDS
to the diluted effluent discharges of a chemical production plant for 3
months. The plant produced different dyes and chemicals and the waste
water therefore could be contaminated with a variety of aliphatic and
aromatic amines and some cyclic aromatic hydrocarbons. After termination
of the treatment, liver and gill DNA from exposed and control trouts was
analysed by [ 32 P]postlabelling for the presence of DNA adducts.
The DNA was enzymatically hydrolysed to the nucleotides. Adducted
nucleotides were extracted with butanol in the presence of the phase
transfer agent tetrabutylammonium chloride and postradiolabelled with
[ 32 P]ATP and PNK. The labelled nucleotides were separated by
multidirectional TLC with 1.0 M phosphate buffer, pH 6.6, in D1, 0.4 M
ammonia in D3 and 4 N ammonia/propanol (1.2:1) in D4. A final
development in direction D4 with 1.0 M phosphate buffer, pH 6.6, was
used as background clean up.
In the trouts exposed to control water no DNA adducts were detectable,
neither in the livers nor in the gills (Figure 6.7). In contrast, in the trouts
exposed to the highest concentration of the waste water, at least 4 DNA
adducts could be found in the livers and in the gills. The overall DNA
adduct level in the exposed trouts was relatively low (1 adduct per 10 8
nucleotides, which indicated only a minimal cancer risk for the exposed
fish.
Limitations
However, the methods presented for adduct determination have their
limitations. For protein adduct determination the most popular method is
by HPLC with electrochemical or fluorescence detection after hydrolysis
and extraction of the adducts. This is due to the low cost and time
consumption of the method. This method is hampered by the possibility of
interferences, which can elute in the range of the compounds of interest. For
an exclusion of haemoglobin adducts formation at low levels it is therefore
crucial to obtain additional information about the chromatographic peaks
of interest, such as for example, by GC/MS.
DNA adducts are often assessed by [ 32 P]postlabelling. This method is
limited by low yields of the enrichment and labelling procedures and by
choosing the appropriate chromatographic conditions for the resolution of
the labelled adducts. The lack of detectability of some DNA adducts,
although they may contain aromatic moieties, enforces the use of a positive
standard in order to check for the yield of the enrichment and the labelling
reaction, and to check for appropriate chromatographic conditions to
resolve the adducts.