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 chromosome damage and sister chromatid exchanges in heroin addicts, particularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evidence of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed graded, 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 potent mutagen, ethylmethanesulfonate (EMS). There were also dose-dependent increases in the lengths and densities of the comet tails observed. These findings were confirmed by a series of MGE experiments in which several days of morphine exposure preceded a 2-hr pulse of EMS. Second, mutant frequency (MF) assays also indicated significant opiate effects. These studies required separate assessment of cloning efficiencies and the frequencies of TG-resistant, HPRT-deficient mutant clones under four test conditions: no treatment, morphine alone for 4 days, morphine plus EMS, and EMS alone. Prior to the treatment phase, aminopterin was used to eliminate background HPRT mutations. The medium was changed after the treatment phase, the cells were allowed to express mutant phenotypes, and then TG was added and resistant mutant clones counted 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-fold to 5.1 x 10(-6) and 7.0 x 10(-6), respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Preliminary analysis with the single strand conformational polymorphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirmed the occurrence of Exon 3 mutants of the HPRT gene in cells exposed to morphine plus EMS. It appears that brief EMS exposure can be repaired, whereas, if morphine exposure persists through one or more cell cycles, direct or indirect mutagenesis is initiated.
Shafer
D A
DA
Human and Behavioral Genetics Research Lab, Georgia Mental Health Institute, Atlanta 30306.
Xie
Y
Y
Falek
A
A
D000704:Analysis of Variance; D001483:Base Sequence; D002478:Cells, Cultured / Q000187:drug effects; D016009:Chi-Square Distribution; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000032:analysis; D004357:Drug Synergism; D004587:Electrophoresis, Agar Gel; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D016015:Logistic Models; D008969:Molecular Sequence Data; D009020:Morphine / Q000633:toxicity*; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D009153:Mutagens; D009020:Morphine; D005020:Ethyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase