DETECTION OF OPIATE-ENHANCED INCREASES IN DNA-DAMAGE, HPRT MUTANTS, AND THE MUTATION FREQUENCY IN HUMAN HUT-78 CELLS

In previous studies we have shown highly significant increases in chro mosome damage and sister chromatid exchanges in heroin addicts, partic ularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evide nce of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed grad ed, dose-dependent, significant increases (P <.0001) in the frequency of comet tails of fragmented DNA when cells were treated with morphine alone (5 x 10(-9)M up to 10(-7)M) or when co-treated with the more po tent mutagen, ethylmethanesulfonate (EMS). There were also dose-depend ent increases in the lengths and densities of the comet tails observed . These findings were confirmed by a series of MGE experiments in whic h several days of morphine exposure preceded a 2-hr pulse of EMS. Seco nd, mutant frequency (MF) assays also indicated significant opiate eff ects. These studies required separate assessment of cloning efficienci es and the frequencies of TG-resistant, HPRT-deficient mutant clones u nder four test conditions: no treatment, morphine alone for 4 days, mo rphine plus EMS, and EMS alone. Prior to the treatment phase, aminopte rin was used to eliminate background HPRT mutations. The medium was ch anged after the treatment phase, the cells were allowed to express mut ant phenotypes, and then TG was added and resistant mutant clones coun ted after 16 days. The background MF level for controls and for cells treated with EMS alone were negligible at 5.12 x 10(-8) and 7.25 x 10( -8) respectively. In the cells treated with morphine alone or morphine plus EMS, MF levels increased very significantly (P <.001) by >100-fo ld to 5.1 x 10(-6) and 7.0 x 10(-6), respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Pr eliminary analysis with the single strand conformational polymerphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirme d the occurrence of Exon 3 mutants of the HPRT gene in cells exposed t o morphine plus EMS. It appears that brief EMS exposure can be repaire d, whereas, if morphine exposure persists through one or more cell cyc les, direct or indirect mutagenesis is initiated. (C) 1994 Wiley-Liss, Inc.