CYTOGENETIC AND IMMUNOFLUORESCENCE ANALYSIS OF BENZO[A]PYRENE-DNA ADDUCT FORMATION AND CHROMOSOME-DAMAGE IN LARVAL BRAIN NEUROBLASTS OF DROSOPHILA-MELANOGASTER

Recently we have evaluated the relationship between benzo[alpha]-pyren e(BaP)-DNA adducts, determined by P-32-postlabelling, and clone freque ncies in the somatic mutation and recombination test (SMART) in Drosop hila melanogaster. Following that study we proceeded to characterise f urther the mechanism of induction of genetic damage in vivo by BaP in Drosophila by cytogenetic analysis of larval brain neuroblasts. Third stage larvae were treated with 4 and 10 mM BaP for 24, 48 or 72 h, In all cases, the larvae were killed 72 h after the beginning of treatmen t, entailing 48, 24 or 0 h post-treatment recovery in BaP-free medium, respectively, At the end of the treatment the following data were col lected: (i) the types and levels of chromosome aberrations in neurobla st metaphase and anaphase nuclei; (ii) the distribution and level of B aP-DNA adducts, revealed by indirect immunofluorescence in neuroblast nuclei using an anti-(BaP-DNA) antibody. The results indicate that BaP induces chromosome breaks, deletions and exchanges in this system. In particular, chromosome exchanges decrease as the post-treatment recov ery time increases, and the dynamics of breaks and deletions appear to be inversely related to those of the exchanges. This suggests that ex changes may require few preconditions to occur and are thus expressed soon after treatment. Chromosome breaks and deletions could require mu ltiple single events before the actual damage is expressed (even some cell divisions away from the end of treatment). The immunoffuorescence analysis suggests that BaP-DNA adducts are more abundant in the heter ochromatin of the neuroblast nuclei. The number of nuclei with a fluor escent signal localised outside the heterochromatic areas decreased as the recovery time of the larvae in normal medium increased, suggestin g that BaP-DNA adducts are removed more rapidly in euchromatin than in heterochromatin.