Expression and methylation analysis of p15 and p16 in mouse bone marrow cells exposed to 1,4-benzoquinone

Abstract

Benzene is an important industrial chemical. It is also an environmental pollutant recognized as a human carcinogen. Both prenatal and adult exposures to benzene are associated with the development of leukemia. To understand the mechanism of benzene-induced epigenetic variations, we investigated the expression and methylation patterns of CpG (phosphodiester bond between cytosine and guanine) islands in p15 and p16 promoter regions in 1,4-benzoquinone (1,4-BQ)-treated primary cultivated C57BL/6J mouse bone marrow cells in vitro. The cell toxicity of 1,4-BQ was evaluated by cell viability test, real-time PCR was used to measure the mRNA expression levels, and bisulfite sequencing PCR (BSP) was used to look into the methylation patterns. The cell viability test indicates that 1,4-BQ exhibited a dose-dependent toxicity to mouse bone marrow cells. After a 24-h exposure to 1,4-BQ at final concentrations of 0, 0.1, 1, and 10 μmol/L, the mRNA expression of p15 and p16 decreased with the increase in 1,4-BQ concentration. The BSP results gathered from the exposure and the control groups were the same. In summary, despite the observation that short-term exposure to 1,4-BQ primary cultivated mouse bone marrow cells decreased the p15 and p16 transcripts, with no influence by their gene promoter methylation.

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

“…The concentration-dependent cytotoxic effect of 1,4-BQ on mouse BM cells observed in our study is in agreement with the results of a previous study, in which mouse BM cells were exposed to 1,4-BQ at concentrations ranging from 0 to 40 μmol/L for 24 h (Tian et al, 2012). Further analysis of the mode of cell death for 1,4-BQ-exposed mouse BM cells revealed a remarkable induction of apoptosis rather than necrosis.…”

“…Mouse BM cells were obtained through femur and tibia flushing (Green et al, 1981;Gaido and Wierda, 1984;Yoon et al, 2003;Tian et al, 2012) from male ICR (Imprinting Control Region)-strain mice, which purchased from Laboratory Animal Resource Unit (LARU), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. All procedures involving the use of laboratory animals were reviewed and were approved by the Animal Ethics Committee of Universiti Kebangsaan Malaysia (UKMAEC, Kuala Lumpur, Malaysia; approval number: FSKB/BIOMED/2011/ZARIYANTEY/21-JULY/384-JULY-2011-MAY-2014-AR-CAT2).…”

“…Two hundred microliters of mouse BM cell suspension containing 1 × 10 6 cells/mL was added per well to a 96-well culture plate and then treated with 1,4-BQ at a series of concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. The concentrations of 1,4-BQ were selected based on a previous study by Tian et al (2012), where the highest concentration used for cytotoxicity testing was 40 μmol/L. Then, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated further for 4 h at 37°C in a 5% CO 2 incubator.…”

“…1,4-BQ is a highly reactive benzene metabolite that can bind to cellular macromolecules such as protein and DNA (Snyder, 2007). 1,4-BQ has been shown to cause cytotoxicity and DNA damage in previous in vitro studies in human CD34 + hematopoietic stem cells (HSCs) , mouse Lin-Sca-1 + c-kit + HSCs , and murine BM cells (Tian et al, 2012). The ability of benzene to alter the genomic profile of exposed HSCs and hematopoietic progenitor cells (HPCs) could suggest a potential mechanism underlying benzene-induced hematopoietic disorders and malignancies (Smith, 2010).…”

Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1

“…In addition to being behaviorally aversive, pBQN is toxic to mammalian cells (Crespo et al, 2011;Fisher et al, 2011;Sarma et al, 2011;Tian et al, 2012). In humans, prolonged exposure to insect secretions of benzoquinone results in ulcers, blisters or discolored skin lesions with a local burning sensation (de Capitani et al, 2011), but the potential involvement of TRPA1 in these symptoms is unknown.…”