Investigation of mechanism(s) of DNA damage induced by 4-monochlorobiphenyl (PCB3) metabolites

Abstract

Abstract4-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, MPOindependent 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 © 2009 Elsevier Ltd. All rights reserved.Corresponding Author: Dr. Gabriele Ludewig, The University of Iowa, Department of Occupational and Environmental Health, 100 Oakdale Campus #124 IREH, Iowa City, IA 52242-5000 Phone: (319) 335-4650, Fax: (319) 335-4290, gabriele-ludewig@uiowa.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our...

Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts

“…DDR signaling involves a large number of proteins that act as sensors, mediators, transducers and effector proteins [41,42]. Recruitment of the DDR protein γ-H2AX, BRCA1, and 53BP1 to DNA DSBs is a key event in the DDR [43-45]. Defective recruitment of repair factors at DNA DSBs, such as delay in foci assembly and disassembly, is associated with defective DDR.…”

Metabolism and metabolites of polychlorinated biphenyls

“…Although for a long time PCBs were thought to be strictly promoting carcinogens, a lower chlorinated congener (PCB3) was shown to induced point mutations in rat livers in vivo (Lehmann et al, 2007), and, in agreement with this, recent evidence suggests that particularly the lower-chlorinated congeners can be oxidized to genotoxic metabolites, such as arene oxides and quinone species (Espandiari et al, 2003, Espandiari et al, 2004, Robertson and Ludewig, 2011). In fact, the quinone metabolites of PCB3 increase gene mutations in vitro at low micromolar concentrations (Zettner et al, 2007), induce strand breaks (Xie et al, 2010), bind to and inhibit the nuclear protein topoisomerase II (Bender et al, 2006, Bender and Osheroff, 2007, Srinivasan et al, 2001, Srinivasan et al, 2002) and reduce telomerase activity resulting in shortened chromosomal telomeres in cells in culture (Jacobus et al, 2008). Interestingly, only the para -dihydroxy metabolite of PCB3 induced polyploidization and only the ortho -dihydroxy metabolite caused sister chromatid exchanges (Flor and Ludewig, 2010) indicating a binding to other, so far unidentified cellular proteins, as mediators of these genotoxic effects.…”

“…As described above, the chlorination pattern of a PCB congener determines the substrate specificity for CYP forms which in turn my influence organ and cell-specific formation of oxidized metabolites. In addition, cells with specific oxidizing enzymes, like myeloperoxidase in bone marrow cells, may be most likely to produce reactive quinone metabolites and therefore more likely to form DNA and protein adduct (Xie et al, 2010). Understanding of these specific enzyme requirements may elucidate the mechanism of the observed organ-specific carcinogenicity of 34 PCBs.…”

Oxidative DNA damages by chemical exposures at work

“…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 glutathione (GSH) depletion [10]. All the phenolic compounds were able to oxidize DNA bases in human lymphocytes, and also observed that pyrimidine bases were more strongly oxidized in comparison to purine ones.…”