Increased Mitochondrial Mass and Mitochondrial DNA Copy Number Are Biomarkers for Benzene Exposure in Hematopoietic Tissue and Leukemia Cells.

Exposure to benzene and its metabolites increases risk of marrow failure disorders, leukemia and other hematological diseases. However, biomarkers of benzene toxicity have not been comprehensively studied in hematopoietic cells and leukemia cells. We previously reported that benzene metabolites may impair electron chain transport and mitochondrial function (3^rd WHO Conference on Children's Health and the Environment, 7-10 June 2009). Therefore, we hypothesized that alterations in mitochondrial mass and mitochondrial DNA (mtDNA) may occur in bone marrow cells and leukemia cells after benzene exposure to compensate for damaged mitochondria.

Total bone marrow cells from healthy individuals and leukemia cell lines (THP-1, Kasumi-1, K562, Molt-4 and HL-60) were cultured in RPMI media containing 10% fetal bovine serum for 5 days. Benzene was added in cell culture media with 0, 1 and 10mM concentration at 24 hour interval. Cell count was performed using an automated blood cell analyzer (ADVIA120, Siemens, Germany). Viability and apoptosis were assessed by tryptophan blue dye exclusion test and flowcytometry based annexin V staining protocol. Hydrogen peroxide content is measured using the commercial kit (Bioxytech^® H₂O₂-560^TM, OXIS International) according to the manufacturer's instructions. Mitochondrial mass, membrane potential and mtDNA copy number were measured using MitoTracker Green, MitoTracker Red probes (Invitrogen), and real time PCR using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research), respectively.

The number of cells were gradually increased regardless of concentration of benzene in day 3, and then steadily maintained during 3 weeks culture. Interestingly, the growth of K562 cells showed no growth inhibition effect (three fold increase) after 5-day exposure to benzene. Overall viability of five leukemia cell lines disclosed significant decrease after two week treatment of benzene (about 60% of viability was observed in 3- week suspension culture). The proportion of apoptosis was increased in time and dose dependent manner after 2-week treatment of benzene. Interestingly, mitochondrial contents and membrane potentials were dramatically increased in 3-week suspension culture after benzene exposure at dose dependent manner. The level of hydrogen peroxide significantly elevated after two week treatment of benzene (4.4 ± 1.9 μM/mg protein) compared with non-benzene treatment group (1.2 ± 1.0 μM/mg protein; P = 0.001). The average mtDNA copy number was gradually increased after exposure to benzene.

Benzene exposure caused increased mitochondrial mass and mtDNA copy number in response to oxidative stress induced by benzene. So, these mitochondrial changes can be used for biomarkers of benzene toxicity in hematopoietic tissue and leukemia cell.

No relevant conflicts of interest to declare.