Diet-induced obesity modulates epigenetic responses to ionizing radiation in mice

Abstract

Both exposure to ionizing radiation and obesity have been associated with various pathologies including cancer. There is a crucial need in better understanding the interactions between ionizing radiation effects (especially at low doses) and other risk factors, such as obesity. In order to evaluate radiation responses in obese animals, C3H and C57BL/6J mice fed a control normal fat or a high fat (HF) diet were exposed to fractionated doses of X-rays (0.75 Gy ×4). Bone marrow micronucleus assays did not suggest a modulation of radiation-induced genotoxicity by HF diet. Using MSP, we observed that the promoters of p16 and Dapk genes were methylated in the livers of C57BL/6J mice fed a HF diet (irradiated and non-irradiated); Mgmt promoter was methylated in irradiated and/or HF diet-fed mice. In addition, methylation PCR arrays identified Ep300 and Socs1 (whose promoters exhibited higher methylation levels in non-irradiated HF diet-fed mice) as potential targets for further studies. We then compared microRNA regulations after radiation exposure in the livers of C57BL/6J mice fed a normal or an HF diet, using microRNA arrays. Interestingly, radiation-triggered microRNA regulations observed in normal mice were not observed in obese mice. miR-466e was upregulated in non-irradiated obese mice. In vitro free fatty acid (palmitic acid, oleic acid) administration sensitized AML12 mouse liver cells to ionizing radiation, but the inhibition of miR-466e counteracted this radio-sensitization, suggesting that the modulation of radiation responses by diet-induced obesity might involve miR-466e expression. All together, our results suggested the existence of dietary effects on radiation responses (especially epigenetic regulations) in mice, possibly in relationship with obesity-induced chronic oxidative stress.

Figure 1. Bone marrow micronucleus test.

A and C: Frequency of micronuclei in the bone marrow cells of C57BL/6J (A) and C3H (C) mice. B and D: ratio of polychromatic erythrocytes to normochromatic erythrocytes (P/N ratio) in the bone marrow of C57BL/6J (B) and C3H (D) mice. *p<0.05 compared to non-irradiated control (for irradiated mice fed a normal diet) or obese mice (for irradiated mice fed a HF diet).

Figure 2. Cell cycle repartition of bone marrow TER-119⁺ nucleated cells.

TER-119 was used as a lineage marker for erythroid cells. Analysis was performed in C57BL/6J (A) and C3H (B) mice using ModFit LT software. Results are the average of three independent experiments. Error bars represent standard deviation. *p<0.05 compared to control.

Figure 3. Gene promoter methylation in C57BL/6J mouse livers.

A: Heatmap representing the promoter methylation status for 24 liver cancer-related genes in the livers of non-irradiated and irradiated C57BL/6J mice fed a normal or HF diet. B: Representative gels showing the methylation-specific PCR analysis of the promoter methylation status for p16, Mgmt and Dapk genes.

Figure 4. Expression levels of miR-466e-3p target genes.

Expression levels of Zfp704, Cnot7 and Cplx2, relative to control, were measured by quantitative real-time PCR. Results are the average of three independent experiments. Error bars represent standard deviation.

Figure 5. ROS levels in AML12 cells.

ROS levels were measured after treatment with free fatty acids (FFAs) oleic acid and palmitic acid and with miR-466e inhibitor. Results are the average of three independent experiments. Error bars represent standard deviation. Asterisks denote significant differences (t-test, *p<0.01).

Figure 6. Dose-response curves for clonogenic survival of AML12 cells.

AML12 cells were treated for 24 hours with free fatty acids (FFAs: oleic acid, palmitic acid) alone (circles, dotted line) or both with miR-466e inhibitor and FFAs (triangles, continuous line) then exposed to various doses of ionizing radiation. Lipofectamine alone was added to control cells (squares, continuous line). Lines represented fitted curves according to linear quadratic regression. The red dotted line indicates 5% clonogenic survival (DL95). Results are the average of at least three independent experiments. Error bars represent standard deviation. Statistical significance of the difference between dose-response curves was performed using one-way Analysis of Variance (one-way ANOVA) with Bonferroni correction for pairwise group comparisons. *p<0.01 compared to control.