A mechanism for 1,4-Benzoquinone-induced genotoxicity

Abstract

Benzene is a common environmental toxin and its metabolite, 1-4-Benzoquinone (BQ) causes hematopoietic cancers like myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). BQ has not been comprehensively assessed for its impact on genome maintenance, limiting our understanding of the true health risks associated with benzene exposure and our ability to identify people with increased sensitivity to this genotoxin. Here we analyze the impact BQ exposure has on wild type and DNA repair-defective mouse embryonic stem (ES) cells and wild type human cells. We find that double strand break (DSB) repair and replication fork maintenance pathways including homologous recombination (HR) and Fanconi anemia (FA) suppress BQ toxicity. BQ-induced damage efficiently stalls replication forks, yet poorly induces ATR/DNA-PKCS responses. Furthermore, the pattern of BQ-induced γH2AX and 53BP1foci is consistent with the formation of poly(ADP-ribose) polymerase 1 (PARP1)-stabilized regressed replication forks. At a biochemical level, BQ inhibited topoisomerase 1 (topo1)-mediated DNA ligation and nicking in vitro; thus providing mechanism for the cellular phenotype. These data are consistent with a model that proposes BQ interferes with type I topoisomerase's ability to maintain replication fork restart and progression leading to chromosomal instability that has the potential to cause hematopoietic cancers like MDS and AML.

Keywords: Fanconi anemia; double strand break repair; replication fork maintenance; type 1 topoisomerase.

Figure 1. The genotoxic profile that compares the survival fraction of mutant ES cells to their parental controls at 10% (black bar) and at 1% (grey bar) cell survival

A. Exposure to 1,4-Benzoquinone (BQ). B. Exposure to etoposide (ETO). C. Exposure to camptothecin (CPT).

Figure 2. Evaluation of chromosome damage in metaphase spreads (MPS) after ES cells were exposed to BQ, ETO and CPT

A. Images of H2ax^−/− cells exposed to 1) nothing, 2) BQ, 3) CPT and 4) ETO. Arrowheads point to chromosomal abnormalities. Enlarged representative examples of chromosomes include 5) normal, 6) isochromatid break (ICB), 7) chromatid break (CB), 8) radial and 9) extrapericentromeres and telomeres (EPT). B–E. The survival fraction (%SF) is shown on the left panel and the # of chromosomal defects is shown on the right panel. (B) Wild type and Ercc1-mutant IB10 cells. (C) Wild type and Ku70-mutant J1 cells. (D) Wild type and H2ax- and Brca1-mutated TC1 cells. (E) Wild type and Fancb-mutated AB2.2 cells. Cells were also exposed to an equivalently toxic dose of Mitomycin C (MMC), a crosslinking agent that is known to be very toxic to FA-defective cells. The concentration for CPT (100 nM, 16 hours) and MMC (30 nM, 16 hours) results in ~ 10% and 90% survival for control cells and ~ 10% and <0.001% survival for Fancb^Δex2 cells as previously reported [38]. Note that MMC induces a much larger level of cell death and radials relative to control cells than the other agents relative to control cells demonstrating that Fancb^Δex2 cells are particularly susceptible to MMC as compared to the other agents. The total number of MPS observed for each bar and statistics are shown in supplemental tables 1 and 2, respectively.

Figure 3. Fiber analysis

A-C. ES cells were exposed to IdU for 20 minutes and then agent (BQ or HU) for 90 minutes and then CldU for 20 minutes. (A) Percent survival fraction (%SF) using the identical condition as the fiber analysis. (B) Fiber analysis in wild type AB2.2 cells. (C) Fiber analysis in Fancb-mutant cells. D-E. ES cells were exposed to IdU for 30 minutes and CldU + agent for 30 minutes. (D) Percent survival fraction (%SF) using the identical conditions for the fiber analysis. E. Fiber analysis in wild type AB2.2 cells. F. Fiber analysis in Fancb-mutant cells. The total number of fibers observed for each bar and statistics are shown in supplemental tables S3 and S4, respectively.

Figure 4. The purification of γH2AX and RPA at the nascent replication strand using iPOND

A. The percent survival fraction (%SF) using the same condition as for iPOND. B. Western blot to evaluate the protein concentrations at purified nascent replication strands. C-E. Graphs that depict the quantitation of 3 Western blots for (C) γH2AX, (D) RPA pS33 and (E) RPA pS4/S8. Error bars are shown for the average of 3 experiments.

Figure 5. Evaluation of γH2AX and 53BP1 foci in HeLa cells exposed to BQ

A. The formation of a regressed fork (chicken foot). The red asterix is a symbol for DNA damage that stalls a fork. This damage could be a CPT-type 1 topoisomerase cleavage complex. B. Representative examples of nuclei with no foci, colocalized foci, γH2AX foci, and 53BP1 foci. C. Survival fraction after exposure to ionizing γ-radiation [IR: 1-10 Gray (Gy)], olaparib (OLA, 10 μM), BQ, ETO and CPT. D. The percentage of nuclei with separated or colocalized γH2AX and 53BP1 foci. Ten or more foci are needed to be positive. The total number of nuclei observed for each bar and statistics are shown in supplementary tables S5 and S6, respectively.

Figure 6. BQ inhibits type 1 topoisomerase (topo 1)

CPT is a positive control and ETO is a negative control. The relaxed DNA shown in lane 19 is a control that came with the kit.