title: Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts.
abstract: To refine previous studies of chromosome damage (CD) and sister-chromatid exchanges (SCE) in heroin addicts, we applied new methods developed in our laboratory to enhance detection of the cytogenetic effects of low-level radiation exposure in hospital workers. For CD analysis, we applied our thymidine-fluorodeoxyuridine-caffeine (TFC) enhancement procedure in which cells at setup receive 1 x 10(-7) M fluorodeoxyuridine to inhibit thymidylate synthetase and 4 X 10(-5) M thymidine to satisfy the induced requirement, and then in G2 receive 2.2 mM caffeine to modulate DNA repair. For SCE enhancement, caffeine treatment was initiated in G1 at 19 h before harvest. Using both standard and enhanced procedures for CD and SCE analysis, blood samples were evaluated from 20 street heroin addicts and 22 controls. Standard 2-day CD and 3-day SCE assays showed small, insignificant genotoxic increases in addicts while the enhanced CD and SCE assays showed highly significant increases. Most CD events were in the form of chromatid and chromosome breaks. There were no rings and only a few dicentrics were observed in the TFC-enhanced cultures. Although quadriradials are rare, 10 were found in addict TFC-cultures and 3 in control TFC-cultures. With the standard CD assay, the mean number of chromosome breaks per 100 cells was 0.727 for controls and 1.056 for addicts (not significant). With the TFC-enhanced assay, the same measure showed 1.483 chromosome breaks for controls and 5.143 for addicts (highly significant, ANOVA: p less than 0.0001). A highly significant difference was also observed for chromatid-type damage with the TFC-enhanced assay (chromatid breaks per 100 cells: 16.793 for controls; 48.191 for addicts). The SCE data also showed significant differences with the enhanced assay. Scoring 25 cells/condition, standard SCE cultures showed 10.892 SCE/cell for controls and 11.732 SCE/cell for addicts (not significant). With CAF enhancement there were 13.08 SCE/cell for controls and 17.05 SCE/cell for addicts (ANOVA: p less than 0.008). These findings indicate that detection of CD and SCE effects can be significantly enhanced by the use of these new procedures. The finding of greatly increased chromatid damage in the addicts with the TFC procedure suggests that at least part of the CD detected occurred in vitro and is not a product of prior in vivo damage. Therefore exposure to this drug and perhaps other environmental agents may not only leave a residue of DNA or chromosome damage but may also induce a sensitivity to further genotoxic damage that is revealed by using the enhanced procedures.
authors.affiliation: Department of Psychiatry, Emory University School of Medicine, Georgia Mental Health Institute, Atlanta 30306.
authors.forename: D A
authors.initials: DA
authors.lastname: Shafer
authors.forename: V G
authors.initials: VG
authors.lastname: Dunbar
authors.forename: A
authors.initials: A
authors.lastname: Falek
authors.forename: R M
authors.initials: RM
authors.lastname: Donahoe
authors.forename: J J
authors.initials: JJ
authors.lastname: Madden
authors.forename: P J
authors.initials: PJ
authors.lastname: Bokos
mesh_terms: D000704:Analysis of Variance; D002478:Cells, Cultured; D002869:Chromosome Aberrations; D004249:DNA Damage; D006556:Heroin Dependence / Q000097:blood / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000473:pathology; D012016:Reference Values; D012854:Sister Chromatid Exchange