Hydrogen peroxide and sodium fluoride at specific conditions induce recurrent breakages at FRA16D in human lymphocytes

Abstract

Cancer is a group of genetic diseases characterized by an uncontrolled cellular proliferation and DNA instability due to accumulated mutations in proto-oncogenes and tumor suppressor genes. This genetic instability seen in cancer is also a hallmark of genomic weak regions termed fragile sites (FS). FS are site-specific breaks, gaps or decondensations observed on metaphase chromosomes when lymphocytes are cultured under replication stress. FS are categorized based on their prevalence into rare fragile sites or common fragile sites (CFS) found in all individuals. Some breakages at CFS have been shown to colocalize with cancer breakpoints. Inducers of CFS can be chemical, environmental or biological. Aphidicolin (Aph) a partial inhibitor of DNA polymerases is the main inducer of CFS. Previous studies have shown that chemicals such as hydrogen peroxide (H2O2) and sodium fluoride (NaF) interfere with DNA integrity and lead to chromosomal instability. We propose in this study to test whether H2O2, a byproduct of biochemical pathways, and NaF, a chemical that is regularly consumed by humans, lead to specific breaks at CFS and to determine if they have a synergistic effect with Aph. Lymphocytes from 3 healthy volunteers not affected by cancer were cultured and treated with 8 different conditions of H2O2 with or without Aph: 1 mM-1 hour (hr); 10 mM-1 hr; 5 mM-18 hrs; 10 mM-18 hrs. As for NaF 6 conditions were tested for 24 hrs with or without Aph: 0.5 mg/L, 1 mg/L and 2 mg/L, in addition to 10 mg/L without Aph. Cytogenetic results showed that 1 mM H2O2-1 hr and 5 mM H2O2-18 hrs treatments increased general breaks in 2 donors up to 4.5 times compared to nocodazole. At 10 mM H2O2 -1 hr and 18 hrs, one donor showed a striking 6.5 and 2 times increase in breaks respectively compared to nocodazole. A synergestic effect was noted in one donor at 1 mM H2O2-1 hr and 10 mM H2O2-18 hrs. As for NaF, 2 donors exhibited up to 7 times increase in the percentage of breaks only at 2 mg/L NaF compared to nocodazole and a synergetic effect was seen in a single donor at 1 mg/L NaF. Molecularly, FISH experiments showed an increase of breakage at FRA16D of 5 and 4 times at 5 mM H2O2-18 hrs and 2 mg/L NaF as compared to nocodazole in 1 donor. A synergetic effect was also noted in one donor at 5 mM H2O2-18 hrs compared to Aph. This study is the first of its kind to identify H2O2 and NaF as inducers of CFS, which can increase genomic instability and can lead to cancer. Further studies on other CFS and additional donors must be conducted.