Deoxyribonuclease I is essential for DNA fragmentation induced by gamma radiation in mice

Abstract

Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates gamma-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in gamma-radiation-induced cell death in radiosensitive organs.

FIG. 1

Inhibition of human recombinant DNase I or rat serum DNase (Serum) in the presence of Znchloride, Zn-DIPS or DIPS (0, 100 or 200 μM). C, control non-digested pBR322 plasmid DNA. Circular covalently closed DNA (cccDNA) is converted first to open circular DNA (ocDNA), then to linear DNA (linDNA) and finally is fragmented (frDNA). Endonuclease activity is shown as a percentage of 0 μM samples.

FIG. 2

Amelioration of radiation-induced injury of jejunum in wild-type (WT) and DNase I knockout (KO) mice treated with or without Zn-DIPS. Panel A: Representative images of the radiation-induced DNA fragmentation (TUNEL, green staining) and reduced cell numbers (DAPI, blue color) in villi and crypts. TUNEL-positive cells are present in the epithelium and stroma. Villi of wild-type animals often had interruptions of epithelial line (gaps) at 16 h after irradiation. Scale bar 20 mm. Quantification of TUNEL (panels B, D, F) and cell numbers (panels C, E, G) was performed in the epithelial (panels B, C) and stromal (panels D, E) parts of villi and in the crypt part of the intestine (panels F, G). *P < 0.05 compared to control mice of same genotype, #P < 0.05 compared to irradiated wild-type mice for the same time/treatment, ¶P < 0.05 compared to irradiated mouse of the same genotype at 16 h. n = 6–12 per group.

FIG. 3

Amelioration of radiation-induced injury to ileum in wild-type (WT) and DNase I knockout (KO) mice treated with or without Zn-DIPS. Panel A: Representative images of the radiation-induced DNA fragmentation (TUNEL, green staining) and reduced cell numbers (DAPI, blue color) in villi and crypts. Scale bar 20 μm. Quantification of TUNEL (panels B, D, F) and cell numbers (panels C, E, G) was performed in the epithelial (panels B, C) and stromal (panels D, E) villi and in the crypt part of the intestine (panels F, G). *P < 0.05 compared to control mice of the same genotype, #P < 0.05 compared to irradiated wild-type mice for the same time/treatment, ¶P < 0.05 compared to irradiated mouse of the same genotype at 16 h. n = 6–12 per group.

FIG. 4

Amelioration of radiation-induced injury of the spleen (white pulp) in wild-type (WT) and DNase I knockout (KO) mice with or without Zn-DIPS. Panel A: Representative images of the radiation-induced DNA fragmentation (TUNEL, green staining) and reduced cell number (DAPI, blue color) in white pulp. Scale bar 20 mm. Quantification of TUNEL (panel B) and cell numbers (panel C) in white pulp. *P < 0.05 compared to control mice of same genotype, #P < 0.05 compared to irradiated wild-type mice for the same time/treatment, ¶P < 0.05 compared to irradiated mouse of the same genotype at 16 h. n = 6–12 per group.

FIG. 5

Amelioration of radiation-induced injury in bone marrow from femurs in wild-type (WT) and DNase I knockout (KO) mice treated with or without Zn-DIPS. Panel A: Representative images of the radiation-induced DNA fragmentation (TUNEL, green staining) and reduced cell numbers (DAPI, blue color) in bone marrow. Scale bar 20 μm. Quantification of the TUNEL (panel B) and cell numbers (panel C) in bone marrow. *P < 0.05 compared to control mice of same genotype, #P < 0.05 compared to irradiated wild-type mice for the same time/treatment, ¶P < 0.05 compared to irradiated mouse of the same genotype at 16 h. n = 6–12 per group.

FIG. 6

DNase I has no effect on radiation-induced injury of major DNase I-producing organs: salivary glands (panel A), pancreas (panel B) and kidney (panel C). Scale bars 20 μm.