Toxicology of Industrial Compounds

natural deoxynucleoside-3′-monophosphates (dNp) and the dNp adducts.
The adducted dNp carrying bulky or aromatic substituents are enriched by
extraction with butanol in the presence of a phase transfer agent or by
selective digestion of the natural (unadducted) dNp with nuclease P1. The
enriched adducted dNp are labelled with [ 32 P]- or [ 33 P]ATP (Figure 6.4).
Polynucleotide kinase T4 is used to catalyse the transfer of the labelled
phosphate group from ATP to the 5′ position of the dNp. The labelled
deoxynucleoside-3′,5′-bisphosphates are then separated by multidirectional
TLC on polyethyleneimine coated cellulose. Radioactive impurities and
unused ATP are running to the top with phosphate buffer (D1), whereas
nucleotides carrying aromatic or bulky adducts are retained at or near the
origin. The part containing the impurities and unused ATP is cut off, and
the adducts are chromatographed in D3 (opposite to D1) and D4
(perpendicular to D3) with ammonia and ammonia/ propanol or urea
containing buffers. Adduct spots are visualised and quantified by
autoradiography and Cherenkov counting or by phosphor imaging (Gupta,
1985; Reddy and Randerath, 1986; Beach and Gupta, 1992).
Alternatively, the enriched nucleotide adducts can be chemically
derivatised with fluorescent labels and analysed by HPLC with fluorescence
detection. However, this method does not reach the sensitivity of the
radioactive assay (Sharma and Jain, 1991; Jain and Sharma, 1993).
Comparison of different methods
P.SAGELSDORFF 79
The methods used for the determination of protein and DNA adducts are
summarised in Tables 6.1 and 6.2. Special attention is drawn to the cost of
equipment and time required for analysis.
HPLC methods with electrochemical or fluorescent detection are
relatively insensitive and only applicable with compounds which are
strongly fluorescent or electrochemically active. Since the costs for the
equipment used and the time consumption are relatively low, these
methods are attractive in certain cases. GC with electron capture detection
or GC/MS offers better sensitivity. However, the method requires
derivatisation. In addition, the costs for the equipment of the GC/MS
methods are quite high. Immunoassays are very sensitive, but involve a
number of time consuming steps for the preparation of an appropriate
antibody, and are only possible if the structure of the respective adduct is
known. The postlabelling method for DNA adducts offers the best
sensitivity, with low equipment costs and low to medium time
consumption. However, the standard method only detects bulky or
aromatic adducts.