Detection of opiate‐enhanced increases in DNA damage, HPRT mutants, and the mutation frequency in human HUT‐78 cells

Abstract

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.

Intravenous opioid drug abuse as an independent risk factor for supraglottic squamous cell carcinoma—A case‐control study

“…Chiesara et al found in peripheral lymphocyte DNA a 7.9 times greater incidence of chromosome anomalies in heroin‐marijuana addicts compared to marijuana‐only addicts. Opiates can indirectly increase the frequency of DNA damage and thereby amplify the occurrence of cells carrying mutagenic alterations . The second possible explanation may be related to immune suppression triggered by the opioids.…”

“…Opiates can indirectly increase the frequency of DNA damage and thereby amplify the occurrence of cells carrying mutagenic alterations. 31 The second possible explanation may be related to immune suppression triggered by the opioids. A forensic study of cadaveric thymus tissues showed that bleeding and dystrophic calcifications in the thymocyte's parenchyma were more common among intravenous drug abusers.…”

Role of P38 Mitogen-Activated Protein Kinase Phosphorylation and Fas-Fas Ligand Interaction in Morphine-Induced Macrophage Apoptosis

“…(24,25). Previously, various investigators reported that morphine enhances DNA damage in lymphocytic cells (37)(38)(39). These investigators suggest that opiates might be either decreasing the capacity to repair the damaged DNA or enhancing sensitization for DNA damage (37)(38)(39).…”

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests

“…Since the data from the chromosomal aberration effects in mouse lymphocytes has not been evaluated independently, it is not clear whether morphine is a unique in vivo positive, but this is worthy of further study. Increases in DNA fragmentation (Comet assay) and HPRT mutations have been reported in the human HUT-78 cell line exposed to morphine in vitro [36].…”