Comparison of induction and repair of adducts and of alkali-labile sites in human lymphocytes and granulocytes after exposure to ethylating agents

Abstract

A comparative study has been made of the induction and repair of adducts and alkali-labile sites in the DNA of human lymphocytes and granulocytes exposed to the ethylating agents N-ethyl- N-nitrosourea (ENU) and diethyl sulphate (DES). To evaluate these damages, the human blood cells were treated with highly 3H-labelled ENU and DES, and the resulting 3H-ethyl adducts were analysed via HPLC. Alkali-labile sites introduced in the DNA during treatment with non-radioactive ENU and DES were detected by alkaline elution with fluorometric quantitation of the DNA in the eluted fractions. All known adducts induced by ENU and DES could be detected by the HPLC methods applied. Furthermore, these adducts were separated from a number of unidentified compounds, because of the improved resolution on the columns used. Most of the adducts were rather persistent during a subsequent incubation period of up to 20 h after treatment, but some partly disappeared (7-ethyladenine and 3-ethyladenine). The induction of alkali-labile sites in lymphocytes and granulocytes was very similar, but the kinetics of the removal of these sites appeared to be quite different. In granulocytes there was hardly any repair, whereas in lymphocytes, particularly after ENU treatment, a substantial and relatively fast repair was observed. Induction of alkali-labile sites in human lymphocytes and granulocytes occurred also at 0°C; the data suggest that this kind of damage is not a result of enzymic repair processes. A comparison of the induction and the repair of alkali-labile sites in lymphocytes and granulocytes with those of the various ethyl adducts did not give a clue as to the identity of the adduct that could be responsible for the lability towards alkali.