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Published on: July 3, 2015
Assessment of methyl thiophanate-Cu (II) induced DNA damage in human lymphocytes
Quaiser Saquib1, Abdulaziz A Al-Khedhairy, Saud Al-Arifi
1Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
Abstract:
Dimethyl 4,4'-(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a category-III acute toxicant and suspected carcinogen to humans. Hence, the ability of this benzimidazole class of fungicide to engender DNA strand breaks was investigated using alkaline single cell gel electrophoresis (SCGE), alkaline unwinding and cytokinesis-blocked micronucleus (CBMN) assays. The SCGE of human lymphocytes treated with 1mM MT for 3h at 37 degrees C showed much higher Olive tail moment (OTM) value of 40.3+/-2.6 (p<0.001) vis-à-vis 3.3+/-0.09 in DMSO control. Treatment of cultured lymphocytes for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). Stoichiometric data revealed the intrinsic property of MT to bind with Cu (II) and its reduction to Cu (I), which is known to form reactive oxygen species (ROS). We have detected the intracellular ROS generation in MT treated lymphocytes and observed an elevated level of MT-induced strand breaks per unit of calf thymus DNA in presence of Cu (II). Overall the data suggested that the formation of MT-Cu (II)-DNA ternary complex and consequent ROS generation, owing to Cu (II)/Cu (I) redox cycling in DNA proximity, is responsible for MT-induced DNA damage.
Insights
Methyl thiophanate (MT), a fungicide, causes DNA strand breaks in human cells. This damage is linked to MT
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- Methyl thiophanate (MT) is a benzimidazole fungicide classified as an acute toxicant and suspected human carcinogen.
- Understanding the genotoxic potential of MT is crucial due to its potential health risks.
- Previous studies have not fully elucidated the mechanism of MT-induced DNA damage.
Purpose of the Study:
- To investigate the DNA-damaging effects of methyl thiophanate (MT) in human lymphocytes.
- To elucidate the mechanism underlying MT-induced genotoxicity, including the role of reactive oxygen species (ROS) and metal ion interactions.
- To assess the potential carcinogenic risk associated with MT exposure.
Main Methods:
- Alkaline single cell gel electrophoresis (SCGE) to measure DNA strand breaks.
- Alkaline unwinding assay to quantify DNA damage.
- Cytokinesis-blocked micronucleus (CBMN) assay to detect chromosomal damage and assess nuclear division.
- Spectrophotometric analysis to study MT's interaction with copper ions (Cu(II)).
- Intracellular ROS generation measurement in treated lymphocytes.
Main Results:
- MT treatment significantly increased DNA strand breaks, as indicated by a higher Olive Tail Moment (OTM) in SCGE assays.
- MT induced a dose-dependent increase in micronucleated cells and a reduction in the nuclear division index (NDI) in CBMN assays.
- MT demonstrated an intrinsic ability to bind with Cu(II), leading to Cu(I) formation and subsequent ROS generation.
- Elevated levels of MT-induced DNA strand breaks were observed in the presence of Cu(II), suggesting a role for copper-mediated ROS production.
- Intracellular ROS generation was detected in MT-treated lymphocytes.
Conclusions:
- Methyl thiophanate (MT) is a potent inducer of DNA strand breaks and chromosomal damage in human lymphocytes.
- The genotoxicity of MT is mediated by the formation of an MT-Cu(II)-DNA ternary complex, leading to ROS generation via Cu(II)/Cu(I) redox cycling.
- The findings highlight the potential carcinogenic risk of MT and underscore the importance of understanding its mechanism of action for risk assessment and regulation.
