Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

Abstract

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.

Fig 1. TSA or MCP30 restores the HQ-induced increased HDAC activity, decreased Topo IIα expression, and the resulting apoptosis in human bone marrow mononuclear cells.

(A—C) Mononuclear cells were isolated from bone marrow aspirates from four healthy donors and subsequently treated with or without 100 μM HQ, in the presence or absence of TSA (0.5 μM) or MCP30 (1 μg/ml). After 24 h, the activity of HDAC (A), the expression of Topo IIα (B), and apoptosis (C) were determined using HDAC activity assay kit, western blot, and Annexin V/PI double staining, respectively. NS stands for normal saline. Histone H3 was used as a loading control for nuclear protein in western blot analysis. (D) Human bone marrow mononuclear cells were treated with or without a Topo IIα inhibitor daunorubicin (200 nM) for 48 h and apoptosis was measured using Annexin V staining. Statistical data and representatives of four independent experiments from different healthy donors were shown. *P < 0.05, compared to the respective control group.

Fig 2. TSA or MCP30 restores the expression and activity of Topo IIα in benzene poisoning mice.

Mice inhaled 300 ppm benzene vapor for 8 weeks and TSA or MCP30 was intraperitoneally injected at a dose of 1 mg/kg. After all mice were killed, bone marrow mononuclear cells were separated and measured the expression of Topo IIα including mRNA (A), protein (B), and activity (C) using RT-PCR, western blot, and Topo II activity assay kit, respectively. GAPDH was used as a reference gene in RT-PCR analysis, and TBP was used as loading control in western blot analysis. Images representing 8 mice per group were shown in left column and statistical data were shown in right column. *P < 0.05, compared to the control group; ^#P < 0.05, compared to the benzene alone-treated group.

Fig 3. TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice.

After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 and anti-acetylated histone H4. (A) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. (B) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. (C) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ^**P < 0.01, compared to the control group; ^#P < 0.05, ^##P < 0.01, compared to the benzene alone-treated group.

Fig 4. TSA or MCP30 alters the mRNA levels of regulatory factors in the Topo IIα promoter in benzene poisoning mice.

After all mice were killed, bone marrow mononuclear cells were separated and the mRNA levels of regulatory factors, including SP1 (A), C-MYB (B), SP3 (C), ATF-2 (D), and ICBP90 (E) were determined using RT-PCR. Relative mRNA levels were defined as a ratio of the targeted gene to GAPDH. Representatives of 6 groups with 8 mice each group were shown in left column and statistical data were shown in right column. ^**P < 0.01, compared to the control group. ^#P < 0.05, ^##P < 0.01, compared to the benzene alone-treated group.

Fig 5. TSA or MCP30 relieves the benzene-induced hematotoxicity in benzene poisoning mice.

After the completion of benzene inhalation, peripheral blood was obtained from each mouse by tail vein bleeding and the mice were subsequently killed. (A—C) Benzene alone treatment lowered the level of hemoglobin (Hb), and the number of white blood cells (WBC) and platelet (PLT) counts, and TSA or MCP30 reversed the above indicators in benzene-treated mice. Statistical data of 6 groups with 8 mice each group was shown. ^**P < 0.01, compared to the control group; ^#P < 0.05, ^##P < 0.01, compared to the benzene alone-treated group. (D) The femurs were separated and embedded in paraffin and subsequently cut into slices with a thickness of 5 μm. After hematoxylin and eosin staining, femoral morphology was observed under a light microscope at a magnification of 400. Images shown were representative of at least three mice each group.