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

Abstract

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.

Figure 1

DNA damage induced by PCB3-HQ (A) and PCB3-pQ (B) in HL-60 and Jurkat cells measured by the standard comet assay. The Olive tail moment is used as the metric for DNA damage. Treatments were conducted for 1 h at 37 and 6 °C.

Figure 2

Intracellular level of ROS measured by DCF fluorescence after exposure to PCB3-HQ for 1h and 3 h in HL-60 cells at (A - D) and Jurkat cells (E – H) at 37°C (graphs in left column) and 6°C (graphs in right column). *, significantly different compared to the 1 h control, P<0.05; # significantly different compared to the 3 h control, p<0.05.

Figure 3

MPO activity and protein levels in HL-60 cells, HL-60 cells after 72h incubation with the MPO heme synthesis inhibitor succinylacetone (SA), and in Jurkat cells determined by the guaiacol oxidation assay and western blot analysis (insert), respectively. *, significantly different from HL-60 cells without SA treatment, p<0.05.

Figure 4

MPO activity in HL-60 cells treated for 1 and 3h with PCB3-HQ (A) or PCB3-pQ (B). *, significantly different from the corresponding control, p<0.05.

Figure 5

Intracellular level of ROS in HL-60 cells with and without exposure to 200 μM succinylacetone for 72 h to inhibit MPO followed by exposure to PCB3-HQ for 1h (A) and 3h (B) or PCB3-pQ for 1 (C) and 3h (D). ^a, significantly different compared to normal control cells, p<0.05 ; ^b, significantly different compared to SA-treated controls, p<0.05 ; ^c, significant difference between corresponding normal and SA-pretreated cells, p<0.05.

Figure 6

Intracellular thiol (A,C) and GSH (B,D) status in HL-60 cells and HL-60 cells pretreated with the GSH-depleting DEM after exposure to PCB3-HQ (A,B) or PCB-pQ (C,D). ^a, significantly different compared to normal control cells, p<0.05 ; ^b, significantly different compared to DEM-pretreated controls, p<0.05 ; ^c, significant difference between corresponding normal and DEM-pretreated cells, p<0.05.

Figure 7

Intracellular ROS level in HL-60 cells with and without pretreatment with 1mM DEM for 6 h to deplete cellular GSH followed by exposure to PCB3-HQ for 1h (A) or 3h (B) or PCB3-pQ for 1h (C) or 3h (D). ^a, significantly different compared to normal control cells, p<0.05 ; ^b, significantly different compared to DEM-pretreated controls, p<0.05 ; ^c, significant difference between corresponding normal and DEM-pretreated cells, p<0.05.

Figure 8

DNA damage induced by PCB3-HQ (A) and PCB3-pQ (B) in HL-60 cells determined by the standard comet assay and hOGG1 FLARE comet assay. The Olive tail moment is used as the metric for DNA damage. Treatments were conducted for 1 h at 37°C with and without pre-treatment with succinylacetone (SA) to reduce MPO activity or diethylmaleate (DEM) to lower intracellular GSH levels. * and #, significantly different compared to the control in Comet and Flare assay, respectively, P<0.05.