Superfund Research Program
PCBs: Metabolism, Genotoxicity and Gene Expression in vivo
Project Leader: Larry W. Robertson
Grant Number: P42ES013661
Funding Period: 2006-2020
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4-Monochlorobiphenyl (PCB3) is readily converted by xenobiotic-metabolizing enzymes to dihydroxy-metabolites and quinones. As the research team reported previously, 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 (Zettner et al., 2007). Apparently, each of these two metabolites, which are a redox pair, has a different spectrum of genotoxic effects due to different, metabolite specific mechanisms. Project researchers 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, these scientists 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 ROS and intracellular free –SH groups (monochlorobimane assay, MCB) and free GSH contents (enzyme recycling method) after treatment with PCB3-HQ and PCB3-pQ. They 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.
To further investigate the different modes of action and to gain further insight into the genotoxicity and possible structure-activity relationships, Project scientists measured the effects of the 2-chloro-2`,5`- dihydroxybiphenyl (PCB1-HQ), 3-chloro-2`,5`-dihydroxybiphenyl (PCB2-HQ), 4-chloro-2`,5`- dihydroxybiphenyl (PCB3-HQ), and 4-chloro-3`,4`- dihydroxybiphenyl (PCB3-Cat) on cytotoxicity, sister chromatid exchange (SCE), cellular proliferation and chromosome number. Notably only PCB3-Cat caused a significant increase in SCE levels. Cell cycle progression during exposure, which is indicated indirectly in this assay by the occurrence of metaphases with Harlequin stained chromosomes (cell underwent 2 S-phases) or uniformly dark stained chromosomes (underwent less than 2 S-phases) was inhibited by PCB2-HQ and PCB3-HQ. Most surprising was the finding that up to 96% of metaphases from cells treated with PCB2- or PCB3-HQ were tetraploid, some of which had dark and some Harlequin stained chromosomes. Neither PCB1-HQ nor PCB3-Cat or the negative (solvent) or positive (ethylmethane sulfonate, EMS) control induced this effect. The mechanism of this polyploidization is unknown. Nearly all cancer cells are hyperdiploid and polyploidization followed by uneven chromosome loss are hypothesized as an underlying mechanism of carcinogenesis. Thus different PCB metabolites may induce carcinogenesis by different mechanism, including those characterized by SCE induction or polyploidization. These results suggest that understanding the mechanism and structure-activity relationships of this astonishing activity of certain PCB metabolites is needed before the research team can fully understand the significance of these effects for human health.