Investigation of mechanism(s) of DNA damage induced by 4-monochlorobiphenyl (PCB3) metabolites.
4-Monochlorobiphenyl (PCB3) is readily converted by xenobiotic-metabolizing enzymes to dihydroxy-metabolites and quinones. The PCB3 hydroquinone (PCB3-HQ; 2-(4'-chlorophenyl)-1,4-hydroquinone) induces chromosome loss in Chinese Hamster V79 cells, whereas the para-quinone (PCB3-pQ; 2-(4'-chlorophenyl)-1,4-benzoquinone) very efficiently induces gene mutations and chromosome breaks. Apparently, each of these two metabolites, which are a redox pair, has a different spectrum of genotoxic effects due to different, metabolite-specific mechanisms. We hypothesized that the HQ requires enzymatic activation by peroxidases with the formation of reactive oxygen species (ROS) as the ultimate genotoxin, whereas the pQ reacts directly with nucleophilic sites in DNA and/or proteins. To examine this hypothesis, we employed two cell lines with different myeloperoxidase (MPO) activities, MPO-rich HL-60 and MPO-deficient Jurkat cells, and measured cytotoxicity, DNA damage (COMET assay), MPO activity, intracellular levels of reactive oxygen species (ROS) and intracellular free -SH groups (monochlorobimane assay, MCB) and free GSH contents (enzyme recycling method) after treatment with PCB3-HQ and PCB3-pQ. We also examined the modulation of these effects by normal/low temperature, pre-treatment with an MPO inhibitor (succinylacetone, SA), or GSH depletion. PCB3-p-Q increased intracellular ROS levels and induced DNA damage in both HL-60 and Jurkat cells at 37°C and 6°C, indicating a direct, MPO-independent mode of activity. It also strongly reduced intracellular free -SH groups and GSH levels in normal and GSH-depleted cells. Thus the ROS increase could be caused by reduced protection by GSH or non-enzymatic autoxidation of the resulting PCB3-HQ-GSH adduct. PCB3-HQ did not produce a significant reduction of intracellular GSH in HL-60 cells and reduced intracellular free -SH groups only at the highest concentration tested in GSH depleted cells. Moreover, PCB3-HQ induced DNA damage and ROS production only at 37 °C in HL-60 cells, not at 6 °C or in Jurkat cells at either temperature; no significant DNA damage and ROS production was observed in HL-60 cells at 37 °C if MPO activity was inhibited by SA. These studies show that the effects of PCB3-HQ are enzyme dependent, i.e. PCB3-HQ is oxidized by MPO in HL-60 cells with the generation of ROS and induction of DNA damage. However, this is not the case with the PCB3-pQ, which may produce DNA damage by the reactivity of the quinone with the DNA or nuclear proteins, or possibly by indirectly increasing intracellular ROS levels by GSH depletion. These different modes of action explain not only the different types of genotoxicity observed previously, but also suggest different organ specificity of these genotoxins.
Department of Occupational and Environmental Health, University of Iowa, College of Public Health, Iowa City, Iowa 52242, USA.
Wei
W
Xie
Kai
K
Wang
Larry W
LW
Robertson
Gabriele
G
Ludewig
D001713:Biphenyl Compounds / Q000378:metabolism* / Q000633:toxicity*; D002460:Cell Line; D020552:Comet Assay / Q000379:methods; D004249:DNA Damage; D006801:Humans; D009153:Mutagens / Q000378:metabolism* / Q000633:toxicity*; D009195:Peroxidase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D001713:Biphenyl Compounds; D009153:Mutagens; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D009195:Peroxidase; C006983:4-chlorobiphenyl