Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts

Shafer, David A.; Dunbar, Virginia G.; Falek, Arthur; Donahoe, Robert M.; Madden, John J.; Bokos, Peter J.

doi:10.1016/0165-1161(90)90043-n

Cited by 12 publications

(3 citation statements)

References 26 publications

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“…(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).…”

Section: Discussionmentioning

confidence: 99%

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

Singhal

Bhaskaran

Patel

et al. 2002

The Journal of Immunology

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In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-β promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-β Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-β Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-β and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.

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Section: Discussionmentioning

confidence: 99%

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

Singhal

Bhaskaran

Patel

et al. 2002

The Journal of Immunology

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“…Also, Shafer et al 20 demonstrated by Comet assay that while brief ethyl methansulphonate exposure can be repaired by human cell cultures, if morphine exposures persists through one or more cell cycles, direct or indirect mutagenesis is initiated. Indeed, they had previously showed chromosomal aberrations in heroin addicts 21 …”

Section: Comet Assay: Principles and Applicationsmentioning

confidence: 99%

“…Indeed, they had previously showed chromosomal aberrations in heroin addicts. 21 The alkaline version of the Comet assay has been used to evaluate the possible genotoxicity of 3,4-methylenedioxymethamphetamine (MDMA) in mouse brain cells. 22 Data obtained in our lab indicated that MDMA was able to induce DNA fragmentation and oxidized bases to monoaminergic as well as non-monoaminergic neurons in the nigrostriatal pathway, indicating a final common pathway involving the generation of free radicals and oxidative stress.…”

Section: Comet Assay: Principles and Applicationsmentioning

confidence: 99%

The Comet Assay as a Method of Assessment of Neurotoxicity

Frenzilli

Scarcelli

Fornai

et al. 2006

Annals of the New York Academy of Sciences

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Comet assay is a quick and versatile method for assessing DNA damage in individual cells. It allows the detection of single and double DNA strand breaks, as well as the presence of alkali labile sites. DNA breaks may represent the direct effect of some damaging agent, or they may be intermediates in cellular repair. DNA strand breaks may also come from the action of free radicals generated by oxidative stress processes. The present article summarizes some data from our and other groups underlining the suitability of the Comet assay in assessing neurotoxicity and its potential in evaluating drugs of abuse-related genotoxicity.

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

Shafer¹,

Xie

Falek

1994

Environ and Mol Mutagen

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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.

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Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts

Cited by 12 publications

References 26 publications

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

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

The Comet Assay as a Method of Assessment of Neurotoxicity

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

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