Carnosine attenuates cyclophosphamide-induced bone marrow suppression by reducing oxidative DNA damage

Abstract

Oxidative DNA damage in bone marrow cells is the main side effect of chemotherapy drugs including cyclophosphamide (CTX). However, not all antioxidants are effective in inhibiting oxidative DNA damage. In this study, we report the beneficial effect of carnosine (β-alanyl-l-histidine), a special antioxidant with acrolein-sequestering ability, on CTX-induced bone marrow cell suppression. Our results show that carnosine treatment (100 and 200mg/kg, i.p.) significantly inhibited the generation of reactive oxygen species (ROS) and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), and decreased chromosomal abnormalities in the bone marrow cells of mice treated with CTX (20mg/kg, i.v., 24h). Furthermore, carnosine evidently mitigated CTX-induced G2/M arrest in murine bone marrow cells, accompanied by reduced ratios of p-Chk1/Chk1 and p-p53/p53 as well as decreased p21 expression. In addition, cell apoptosis caused by CTX was also suppressed by carnosine treatment, as assessed by decreased TUNEL-positive cell counts, down-regulated expressions of Bax and Cyt c, and reduced ratios of cleaved Caspase-3/Caspase-3. These results together suggest that carnosine can protect murine bone marrow cells from CTX-induced DNA damage via its antioxidant activity.

Keywords: Apoptosis; Carnosine; Cell cycle arrest; Cyclophosphamide; Oxidative DNA damage; Sister chromatid exchange.

Graphical abstract

Fig. 1

Anti-oxidative effects of carnosine in bone marrow cells of CTX-treated mice. (A) DCFH-DA was applied to analyze the intracellular ROS levels by flow cytometry at 24 h after CTX and carnosine treatment. (B) The relative levels of ROS in bone marrow cells are presented as fold-change compared to control cells. (C) Oxidative DNA levels of bone marrow cells were detected at 1, 5 and 10 days after CTX and carnosine treatment by immunofluorescence detection of 8-oxo-dG. The confocal images were captured at a magnification of 400×, scale bar = 10 µm. (D) The fluorescence intensity of 8-oxo-dG was measured through ImageJ, which are presented as fold-change compared to control cells. Data are expressed as mean ± SD (n = 5). ^##P < 0.01 vs. control group; **P < 0.01, *P < 0.05` vs. CTX group. Car indicates carnosine.

Fig. 2

Carnosine decreased chromosomal abnormality of bone marrow cells in CTX-treated mice. (A) Molecular structure of carnosine. (B) SCE was observed in bone marrow cells by Hoechst-Giemsa staining. Representative images of chromosomal abnormalities are at the same magnification of 1000×. Scale bar = 20 µm. The red arrows indicate SCE. (C) Twenty-five second-division metaphases from each animal were counted and calculated for SCE. Data are expressed as mean ± SD (n = 4). ^##P < 0.01 vs. control group; **P < 0.01 vs. CTX group.

Fig. 3

Carnosine attenuated G2/M cell cycle arrest of bone marrow cells in CTX-treated mice. (A) Diagram of cell cycle analysis in bone marrow cells by flow cytometry assay. (B) Statistical analysis of G1, S, and G2/M populations in bone marrow cells. (C) Western blotting analysis of p-Chk1, Chk1, p-p53, p53 and p21 protein expression. (D) The relative intensity of p-Chk1/Chk1, p-p53/p53 and p21. Data are expressed as mean ± SD (n = 3). ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. CTX group.

Fig. 4

Carnosine suppressed the apoptosis of bone marrow cells in CTX-treated mice. (A) Effects of carnosine on apoptosis in bone marrow cells detected by TUNEL assay. The images of TUNEL positive cells were captured by a microscope at a magnification of 400×, scale bar = 20 µm. (B) Statistical analysis of TUNEL positive rate. (C) Western blotting analysis of Bax, Cyt c, Cleaved Caspase-3 and Caspase-3. (D) The relative intensities of Bax, Cyt c, Cleaved Caspase-3/Caspase-3. Data are expressed as mean ± SD (n = 3). ^##P < 0.01 vs. control group; ^*P < 0.05,^**P < 0.01 vs. CTX group.