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.

Introduction

   Over the last century, ample evidence has been accumulating of a strong
   link between carcinogenesis and exposure to ionizing radiation [31][1].
   The deleterious effects of ionizing radiation are considered to result
   mainly from the direct induction of DNA damage in target cells, but
   also from non-targeted effects (such as radiation-induced genomic
   instability and bystander effect) [32][2]. Ionizing radiation can cause
   chromosomal aberrations, including acentric fragments, which upon cell
   replication are excluded from the main nuclei and form micronuclei (MN)
   [33][3]. The monitoring of MN in erythrocytes of the bone marrow is an
   effective tool to assess the genotoxic effects of ionizing radiation
   even at low levels. Radiation-induced genomic instability is a
   well-documented phenomenon, which can be observed even in the progeny
   of irradiated animals, suggesting the involvement of epigenetic
   mechanisms [34][4], such as the modulation of genome methylation or the
   regulation of micro-RNA expression. Recent data suggest that even low
   dose radiation exposures could also result in epigenetic modifications
   [35][5].

   A major question for radiation biology is to understand how other risk
   factors could influence the biological effects of ionizing radiation,
   especially in terms of cancer risk [36][6], or reversely how ionizing
   radiation exposures could potentialize other risk factors. Obesity and
   associated disorders are increasingly becoming a global health
   challenge. Obesity is a major risk factor for various metabolic
   syndromes (such as insulin resistance, type 2 diabetes and nonalcoholic
   fatty liver disease) [37][7]–[38][9] and for initiation of cancer at
   several organ sites, including breast, endometrium and kidney [39][10].
   Weight gain and obesity was also related to mortality from liver
   cancer, pancreatic cancer, non-Hodgkin lymphoma and myeloma [40][11].
   Nonalcoholic fatty liver disease can evolve into nonalcoholic
   steatohepatitis, which can lead to primary hepatocellular carcinoma
   [41][12]. There is compelling evidence that oxidative stress and
   inflammation are major mechanisms involved in metabolic disorders
   associated with obesity [42][13].

   The susceptibility to develop diet-induced obesity (DIO) and metabolic
   syndromes strongly depends on the genetic background, with heritability
   estimates ranging from 40% to 70% in human populations [43][14],
   [44][15]. Furthermore, a number of studies have shown that various
   mouse strains and their genetic variants show different metabolic
   phenotypes [45][16]. For example, the liver response to a sustained
   high-fat diet was characterized by the activation of peroxisomal
   β-oxidation in C57BL/6J mice and by lipogenesis in 129Sc mice [46][17].
   While C57BL/6J mice exhibited severe obesity and diabetes after being
   fed a high-fat diet, C3H mice fed a similar diet showed normal glucose
   tolerance and no hepatic steatosis even though they exhibited a
   significant weight increase [47][18]. The C57BL/6J mouse strain has
   become a popular experimental model for evaluating the molecular
   mechanisms and consequences of DIO, because they develop insulin
   resistance, hyperglycemia and obesity in a way that closely matches the
   development of human metabolic syndrome [48][19]. In comparison, other
   strains, such as C3H/He, 129/Sv and A/J mice are more resistant to
   diabetes and obesity [49][20].

   Emerging evidence suggests that dietary effects [50][21], as well as
   carcinogenesis [51][22], involve epigenetic mechanisms, such as DNA
   methylation changes or miRNA regulations. On the one hand, aberrant
   gene silencing through promoter hypermethylation has been shown to be a
   crucial event during early neoplastic progression [52][23]. High fat
   diet-induced obesity and metabolic syndromes can also modulate the
   methylation pattern of various gene promoters in human and animal
   models, such as leptin [53][24], peroxisome proliferator activated
   receptor γ (PPARγ) [54][25] or microsomal triglyceride transfer protein
   (Mttp) [55][26]. On the other hand, there is now ample (and still
   growing) data describing cancer-associated miRNA regulations, implying
   the crucial role of miRNAs in maintaining cellular homeostasis
   [56][27].

   While radiation-induced carcinogenesis was significantly reduced in C3H
   mice after calorie restriction [57][28], there is a scarcity of data
   concerning the modulation of radiation responses by high fat diets or
   obesity. In a mouse model of malignant glioma, high fat
   low-carbohydrate ketogenic diet significantly enhanced the
   effectiveness of radiation therapy [58][29]. Obesity also increased
   non-ionizing UV radiation-induced oxidative stress [59][30].

   In this study, we compared the short-term biological responses to
   ionizing radiation in C57BL/6J and C3H mice fed a control normal fat
   diet or a high fat (HF) diet. First, we evaluated whether DIO could
   influence radiation-induced genotoxicity. Then, because liver is one of
   the main target sites for metabolic syndrome, we measured immediate
   epigenetic regulations in relationship with diet and ionizing radiation
   (gene repression through promoter hypermethylation, miRNA regulations).

Materials and Methods

Mice

   14 weeks-old C57BL/6J DIO mice and 5 weeks-old C3H/HeJ mice were
   purchased from Charles River Laboratories (Yokohama, Japan) and SLC,
   Inc. (Hamamatsu, Japan), respectively. C57BL/6J DIO mice are a model of
   pre-diabetic type 2 diabetes with elevated blood glucose and impaired
   glucose tolerance. C3H mice are HF diet-induced obesity resistant. Mice
   were maintained for 3 (C57BL/6J) or 12 weeks (C3H) in a conventional
   animal facility under a 12-h light/12-h dark photoperiod (lights on
   from 7∶00 a.m. to 7∶00 p.m.). Animals were housed in autoclaved cages
   with sterilized wood chips and allowed free access to 10% fat (D12450B,
   Research Diets Inc., New Brunswick, NJ, USA) or 60% fat laboratory chow
   (D12492, Research Diets Inc.) and acidified water (pH = 3.0±0.2). The
   60% fat chow will be referred to as “high fat diet” (HF diet). All
   experimental protocols involving mice were reviewed and approved by The
   Institutional Animal Care and Use Committee of the National Institute
   of Radiological Sciences (NIRS) and were performed in accordance with
   the NIRS Guidelines for the Care and Use of Laboratory Animals.

Cell culture and free fatty acid treatment

   AML12 mouse liver cells (ATCC, CRL-2254) were grown in DMEM/F12 medium
   (Gibco, Gaithersburg, MD, USA) supplemented with 0.005 mg/ml insulin,
   0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone
   (ITS Media Supplement, Sigma-Aldrich, St Louis, MO, USA) and 10% fetal
   bovine serum (FBS, Nichirei Biosciences, Tokyo, Japan). Oleic acid and
   palmitic acid (Sigma-Aldrich) were conjugated to bovine serum albumin
   at a molar ratio of 6∶1. Cells were treated with a mixture of oleic and
   palmitic acid for 24 hours before irradiation.

Irradiation

   Mice were exposed to four times daily doses of 0.75 Gy X-rays (0.25
   Gy/min) generated by a Pantak 320S machine (Shimadzu, Japan) operated
   at 200 kVp and 20 mA, using a 0.50-mm Al +0.50-mm Cu filter. An
   exposure rate meter (AE-1321M, Applied Engineering Inc., Japan) was
   used for the dosimetry. Mice were sacrificed one day after the final
   irradiation.

   AML12 cells were irradiated in serum-free medium 72 hours after
   plating, using an X-ray generator (ISOVOLT Titan-320, General Electric,
   Fairfield, CT, USA) at a dose-rate of 0.9 Gy/min.

Bone marrow micronucleus test

   Diet- and radiation-induced genotoxicity was assessed using the
   standard bone marrow MN test according to the method published by
   Hayashi et al. [60][31]. Bone marrow smears were prepared from both
   femurs. Micronuclei were counted in immature polychromatic erythrocytes
   (PCEs) and in mature normochromatic erythrocytes (NCEs). The
   frequencies of micronuclei in PCEs and NCEs are referred to as MNPCEs
   and MNNCEs, respectively. The ratio of PCEs to mature NCEs (P/N ratio)
   is an indicator of the relative proliferation rate in the erythroid
   lineage, and its decrease is considered to be an indicator of
   mutagen-induced cytotoxicity [61][32]. At least 15000 cells (PCE+NCE)
   per mouse were counted and the data for each experimental point were
   obtained from at least 5 mice.

Flow cytometry

   To identify cells of the erythroid lineage, bone marrow cells were
   incubated immediately after collection, with FITC-conjugated TER-119
   antibody (557915, BD Pharmingen, San Diego, CA, USA) for 20 minutes at
   4°C then washed, fixed with paraformaldehyde/methanol [62][33] and
   resuspended in cold PBS with 3% FBS, 50 µg/mL propidium iodide and 1
   µg/mL RNase. The cell cycle repartition of erythroid cells was then
   analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose,
   CA, USA) with the CellQuest software (Becton Dickinson). Cell cycle
   analysis was performed using ModFit LT software (Verity Software House,
   Topsham, ME, USA).

Methylation PCR array

   For methylation analysis of mouse liver tissues, the EpiTect Methyl
   qPCR profiling service (Filgen, Nagoya, Japan) was used. Genomic DNA
   was extracted from C57BL/6J mouse livers using the QIAmp DNA Mini Kit
   (Qiagen, Chatsworth, CA). The methylation profiling of 22 liver
   cancer-related genes (Ep300, Cdkn1b, Cdkn2a, Fhit, Pycard, Tnfrsf10d,
   Cdh1, Dlc1, Opcml, Reln, Ccnd2, Cdkn1a, Rassf1, Wt1, Socs1, Msh2, Msh3,
   Gstp1, Reln, E2f1, Runx3, Sfrp2) was measured using EpiTect Methyl qPCR
   arrays according to the manufacturer’s instructions (Qiagen). This
   system does not require bisulfite conversion of DNA and relies on
   methylation-sensitive restriction enzymes. Briefly, 1 µg DNA was
   digested overnight at 37°C with the Methyl-Profiler DNA Methylation
   Enzyme Kit (Qiagen) containing methylation-sensitive and
   methylation-specific restriction enzymes, which digest unmethylated and
   methylated DNA, respectively. Then enzymatic activity was inactivated
   at 65°C for 20 min. The remaining DNA in each individual enzyme
   reaction was marked with SYBR Green then quantified using a 7500
   real-time PCR system (Applied Biosystems, Foster City, CA, USA) using
   primers specific to one of the 22 liver cancer-related genes. The
   relative proportions of unmethylated and methylated DNA were then
   measured relative to the total DNA for each gene (measured when no
   restriction enzymes were added).

Methylation-specific PCR

   Genomic DNA was extracted from C57BL/6J mouse livers using the
   NucleoSpin TriPrep kit (Macherey-Nagel, Düren, Germany) according to
   the manufacturer’s instructions. 500 ng DNA was denatured using 0.3M
   NaOH (42°C, 30 min) then bisulfite modification was carried out by
   adding 1020 µL 40.5% sodium bisulfite, 60 µL 10 mM hydroquinone and 10
   µL H[2]O, overlaying the mix with mineral oil and incubating at 55°C
   for 16 hours. The modified DNA was then purified using the Wizard DNA
   cleanup system (Promega) and resuspended in 100 µL TE. The purified DNA
   samples were then denatured (with NaOH) and precipitated with ethanol
   and stored at –20°C until use. The promoter methylation status of p16,
   Mgmt and Dapk genes was determined by methylation-specific PCR using
   primer sets described by Sharpless et al., Yamada et al. and Mittag et
   al, respectively [63][34]–[64][36].

Microarray analysis of miRNA levels

   Total RNA was extracted from the livers of C57BL/6J mice (n = 3 per
   experimental condition) using TRIzol reagent (Invitrogen, Carlsbad, CA,
   USA). The profiling of miRNA expression levels was performed using the
   miRCURY LNA miRNA profiling service (Filgen). The quality of total RNA
   was checked using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA),
   then RNA was labeled with Hy3 and hybridized to miRCURY LNA microRNA
   arrays 7^th generation (Exiqon, Vedbaek, Denmark). Slides were scanned
   using a laser scanner (Molecular Devices, Sunnyvale, CA, USA) and the
   resulting images were analyzed using Array-Pro v.4.5 (Media
   Cybernetics, Bethesda, MD, USA). Following background correction,
   quantile normalization was applied, so that the distribution remains
   the same across arrays [65][37]. Unsupervised hierarchical clustering
   and heatmap vizualisation of the samples for murine probes were
   performed using the CIMminer online software
   ([66]http://discover.nci.nih.gov/cimminer/). Differentially expressed
   miRNA were identified by using the t-test within Significance Analysis
   of Microarrays (SAM) and median normalization [67][38]. The Diana
   miRPath tool v.2.1 [68][39] coupled with microT-CDS database was used
   to determine KEGG pathway [69][40] enrichment within the putative
   targets for the differentially expressed miRNAs. All array data have
   been submitted to the Gene Expression Omnibus (GEO) under the accession
   number [70]GSE47956.

Real-time PCR

   For the validation of methylated genes expression, we used primers for
   Socs1, Ep300, p16, Mgmt and Dapk (QuantiTect Primer Assay, Qiagen); for
   the evaluation of miR-466e-5p gene targets expression, we used primers
   for Zfp704, Cplx2 and Cnot7 [71][41], [72][42]; for the validation of
   miRNA microarrays, we used primers for miR-466e-5p, miR-21-3p and
   miR-185-3p (MystiCq microRNA qPCR Assay Primer, Sigma-Aldrich). Mouse
   liver RNA was reverse-transcribed using the RT2 first strand kit
   (SABiosciences, Frederick, MD, USA) for gene expression experiments and
   MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich) for miRNA
   expression experiments. Then quantitative real-time PCR reactions were
   performed in triplicate with an Applied Biosystems 7300 Real-Time PCR
   system (Life Technologies), using the RT2 SYBR Green PCR Master Mix
   (SABiosciences) for gene expression experiments and the MystiCq
   microRNA SYBR Green qPCR ReadyMix for miRNA expression experiments,
   according to the manufacturers’ instructions.

Measurement of ROS levels

   Intracellular levels of reactive oxygen species (ROS) in AML12 cells
   were measured using
   5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate,
   acetyl ester (CM-H2DCFDA, Molecular Probes, Eugene, OR, USA). Cells
   were plated in 12-well plates and loaded with pre-warmed PBS containing
   a mixture of oleic and palmitic acid for 12 hours, then 10 µM
   CM-H2DCFDA was added and cells were incubated for 40 minutes.
   Fluorescence intensities were measured using a SpectraMax M5 microplate
   reader (Molecular Devices, Sunnyvale, CA, USA) (excitation at 493 nm,
   emission at 520 nm). Unstained cells were used as negative control.

Clonogenic assay

   miR-466e inhibitor (Sigma-Aldrich) was transfected 48 hours before
   irradiation into AML12 cells using Lipofectamine RNAiMax reagent
   (Invitrogen), according to the manufacturer’s instructions
   (lipofectamine alone was added to control cells). Free fatty acids were
   added 24 hours before irradiation. After irradiation, cells treated or
   not with fatty acids were seeded at defined densities and incubated for
   10–14 days then stained. Colonies with >50 cells were scored and
   surviving fractions were determined after correcting for the plating
   efficiency. Survival curve data were fitted to the linear-quadratic
   model [73][43] and are presented as the mean of at least three
   independent experiments.

Results

Diet-induced obesity does not modulate radiation-induced genotoxicity in the
bone marrow

   Pre-diabetic obese C57BL/6J mice fed a high fat (HF) diet for several
   weeks kept gaining weight in our laboratory ([74]Figure S1). C3H mice
   also accumulated additional weight when fed a HF diet, but did not
   exhibit metabolic syndromes. In order to evaluate whether DIO could
   influence radiation-induced genotoxicity in C57BL/6J and C3H mice, we
   measured bone marrow micronuclei frequencies after irradiation
   ([75]Figure 1). Exposure to ionizing radiation resulted in increased
   MNPCE incidence regardless of diet in both mouse strains. No
   significant difference in MNPCE frequencies was observed between obese
   and non- obese animals, suggesting that DIO did not modulate
   radiation-induced genotoxicity.

Figure 1. Bone marrow micronucleus test.

   [76]Figure 1
   [77]Open in a new tab

   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).

   A significant decrease in P/N ratios was observed after irradiation in
   non-obese C57BL/6J and C3H mice ([78]Figure 1B, D), indicative of
   cytotoxic damage or radiation-induced inhibition of proliferation.
   However, while exposure to ionizing radiation did also result in lower
   P/N ratio in obese C3H mice, C57BL/6J did not exhibit a significant
   radiation-induced decrease in P/N ratio. In order to determine whether
   the modulation of P/N ratio involved any modulation of erythroid cell
   proliferation, we studied the cell cycle repartition of nucleated
   erythroid progenitor and precursor cells in the bone marrow ([79]Figure
   2). TER-119+ cells are of the erythroid lineage and represent
   approximately 20% of the total cell number in the bone marrow. No
   significant difference in cell proliferation was observed in C57BL/6J
   mice after irradiation or depending on diet. In C3H mice, erythroid
   progenitors and precursor cells exhibited a lower entry into S/G2/M
   after irradiation or HF diet (p<0.01). The lower percentage of S/G2/M
   cells in irradiated animals suggested that the observed decreased P/N
   ratio resulted from an inhibition of cell proliferation of erythroid
   progenitors and precursors in response to radiation. However,
   surprisingly, C3H mice fed a HF diet did not show decreased numbers of
   erythroid cells in S/G2/M after irradiation, which may suggest that in
   obese mice, the cytotoxic effects of radiation affect mainly PCEs but
   not erythroid progenitors.

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

   Figure 2
   [80]Open in a new tab

   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.

Diet-induced obesity is associated with immediate modulation of promoter
methylation in the liver

   Even though DIO did not apparently modulate radiation-induced
   genotoxicity, we wondered whether immediate effects at the molecular or
   cellular level could be observed, which could ultimately influence
   radiation sensitivity and overall radiation risks in obese mice.
   Because liver is a major target organ for the metabolic syndromes
   resulting from DIO (including fatty liver disease), we then focused our
   investigations on effects in the liver.

   Promoter-specific hypermethylation is a frequent event in liver
   carcinogenesis [81][44]and is dependent on diet [82][45]. For this
   reason, we measured the methylation status of 27 gene promoters in the
   livers of C57BL/6J mice, using methylation PCR arrays and MSP
   ([83]Figure 3). Methylation array provided us with methylation levels
   in the promoters of 24 liver cancer-related genes ([84]Figure 3A). The
   percentages of highly-methylated DNA (as a fraction of total input DNA)
   never exceeded 10% for most of the genes, except E1A binding protein
   p300 (Ep300) and suppressor of cytokine signaling 1 (Socs1). It
   exceeded 20% for Ep300 and Socs1 genes in non-irradiated animals fed a
   HF diet, but it dropped to 7.8% and 12.4%, respectively, after
   irradiation. Our results suggested that Ep300 expression might be
   inhibited in obese mice through promoter methylation, but not after
   irradiation. The results were less clear for Socs1, whose methylation
   levels were already elevated in control animals.

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

   [85]Figure 3
   [86]Open in a new tab

   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.

   Previous investigations have described in various human and animal
   models the epigenetic regulation of promoter methylation for the tumor
   suppressors p16 and Dapk, as well as for the O6-methylguanine-DNA
   methyltransferase Mgmt [87][46]–[88][49]. For this reason, we also
   measured by MSP the methylation status of these three genes in our
   model ([89]Figure 3B). While no methylation was observed in control
   animals, HF diet triggered the methylation of p16, Mgmt and Dapk gene
   promoters (both in irradiated and in non-irradiated animals). Mgmt
   promoter was also methylated after irradiation in mice fed a normal
   diet. Interestingly, expression levels of Ep300, Socs1, p16, Mgmt and
   Dapk in obese and irradiated mice were significantly lower than in
   control mice ([90]Figure 3C).

   Overall our results suggest that the expression levels of several
   anti-tumour genes might be modulated by rapid promoter methylation in
   obese mice, with potential consequences in terms of radiation responses
   or carcinogenic risk (if sustained over the long-term).

Diet-induced obesity influences micro-RNA regulations in the liver after
irradiation

   In addition to the regulation of gene expression by promoter
   methylation, diet-related epigenetic processes can also involve miRNAs.
   miRNA expression profiles in C57BL/6J mouse livers were analyzed by
   using locked nucleic acid (LNA) [91][50] miRNA arrays. Both HF diet and
   irradiation resulted in few consistent miRNA regulations among the
   sampled animals (three animals per experimental group) ([92]Figure S2).
   Therefore SAM analysis was performed to compare miRNA expression levels
   between each experimental group ([93]Table 1). SAM analysis provided a
   limited number of miRNAs that were consistently deregulated in every
   animal of each experimental group. Most of the observed modulations
   were upregulations. When compared to control animals (normal diet,
   non-irradiated), only one miRNA, miR-466e-5p, was upregulated in obese
   mice (no miRNA was downregulated). The putative targets genes of
   miR-466e-5p were identified by miRPath (using microT-CDS database) and
   the Biosynthesis of fatty acids KEGG pathway was identified by pathway
   enrichment analysis (p<0.01). After irradiation, 33 miRNAs were
   upregulated and 1 was downregulated ([94]Table 1). 39 KEGG pathways
   (p<0.01) were found to be altered by miRPath ([95]Table 2), including
   Pathways in cancer, TGF-β signaling, MAPK signaling, Focal adhesion,
   Apoptosis and Wnt signaling pathway, which are all known functions
   associated with radiation exposure [96][51]–[97][53]. On the contrary,
   in irradiated obese mice, only one miRNA (miR-3961) was upregulated
   compared to control and no miRNA was modulated compared to
   non-irradiated obese mice: the above-mentioned radiation-induced miRNA
   regulations were not observed in obese mice. We verified by real-time
   quantitative PCR the expression levels of miR-466e-3p, miR-185-3p and
   miR-21-3p; both miR-466-e-3p and miR-21-3p were upregulated after
   high-fat diet and irradiation, respectively ([98]Table S1). To evaluate
   the biological significance of miR-466e-3p upregulation in obese mice,
   we measured by real-time quantitative PCR the expression levels of
   three target genes: Zfp704, Cnot7 and Cplx2 ([99]Figure 4). These three
   genes were downregulated in mice fed a HF diet.

Table 1. miRNA regulations in the livers of non-irradiated and irradiated
C57BL/6J mice fed a normal or HF diet, determined using Significance Analysis
of Microarrays (SAM).

   HF Irradiated HF, irradiated
   Upregulated Downregulated Upregulated Downregulated Upregulated
   Downregulated
   FC FC FC FC FC FC
   vs Control vs Control vs Control vs Control vs Control vs Control
   miR-466e-5p 1.7 none miR-1187 2.3 miR-5616-5p −2.7 miR-3961 1.5 none
   miR-185-3p 2.7
   miR-1897-5p 2.1 vs HF vs HF
   miR-1900 1.8 none none
   miR-1935 2
   miR-1947-3p 2.3
   miR-1971 2.5
   miR-21-3p 2.9
   miR-3082-5p 2.2
   miR-3098-3p 2.3
   miR-3100-3p 2.5
   miR-32-3p 2.1
   miR-346-3p 2.5
   miR-3961 1.5
   miR-466f 1.9
   miR-466f-3p 2.3
   miR-466i-3p 2.5
   miR-466i-5p 2.2
   miR-466q 2.4
   miR-467e-3p 2.2
   miR-467f 2.5
   miR-467g 2.3
   miR-5113 2.3
   miR-574-5p 2.1
   miR-669a-3-3p 2.2
   miR-669c-5p 2.2
   miR-669e-3p 2.4
   miR-669f-3p 2.4
   miR-669k-5p 1.9
   miR-669l-5p 2.1
   miR-669n 2.1
   miR-669o-5p 1.8
   miR-744-5p 2
   [100]Open in a new tab

   Fold-changes (FCs) of miRNA expression were measured as compared to
   non-irradiated control mice (vs Control) or to non-irradiated mice fed
   a HF diet (vs HF). If the expression ratios were >1, then FCs were
   equal to expression ratios. If the expression ratios were <1, then FCs
   were equal to the opposite of expression ratios.

Table 2. Altered KEGG pathways (p<0.01) associated with the putative target
genes of the miRNAs regulated by ionizing radiation exposure in mice fed a
normal diet.

   KEGG pathway Nb of genes Nb of miRNAs
   1 Pathways in cancer 122 28
   2 TGF-beta signaling pathway 36 23
   3 MAPK signaling pathway 98 29
   4 Focal adhesion 76 27
   5 Glycosaminoglycan biosynthesis - heparan sulfate 7 12
   6 Regulation of actin cytoskeleton 79 27
   7 Colorectal cancer 28 24
   8 Melanoma 34 24
   9 ErbB signaling pathway 34 26
   10 Renal cell carcinoma 30 25
   11 Endometrial cancer 24 24
   12 Apoptosis 32 25
   13 Glioma 25 22
   14 Prostate cancer 38 25
   15 Adherens junction 32 23
   16 Pancreatic cancer 30 25
   17 Calcium signaling pathway 60 27
   18 T cell receptor signaling pathway 43 27
   19 Non-small cell lung cancer 21 22
   20 Melanogenesis 36 24
   21 Long-term depression 23 22
   22 Osteoclast differentiation 44 26
   23 Toxoplasmosis 40 23
   24 Acute myeloid leukemia 24 23
   25 Axon guidance 46 23
   26 Gap junction 33 23
   27 Wnt signaling pathway 55 25
   28 Neurotrophin signaling pathway 48 30
   29 Hedgehog signaling pathway 23 19
   30 Endocrine and other factor-regulated calcium reabsorption 21 21
   31 Lysine degradation 14 16
   32 mTOR signaling pathway 22 21
   33 Ubiquitin mediated proteolysis 45 26
   34 Gastric acid secretion 29 23
   35 SNARE interactions in vesicular transport 14 16
   36 ECM-receptor interaction 25 18
   37 N-Glycan biosynthesis 15 17
   38 Bacterial invasion of epithelial cells 26 26
   39 Glycosphingolipid biosynthesis - lacto and neolacto series 9 15
   [101]Open in a new tab

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

   [102]Figure 4
   [103]Open in a new tab

   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.

Free fatty acids generate oxidative stress and sensitize liver cells to
ionizing radiation

   In order to mimic in vitro the effects of obesity on radiation
   responses in the mouse liver, we treated AML12 murine liver cells with
   free fatty acids (FFAs: palmitic acid and oleic acid) for 12 to 24
   hours. Twelve hours after FFA administration, ROS levels increased
   significantly, reminiscing of chronic oxidative stress in C57BL6 DIO
   mouse livers; the addition of miR-466e inhibitor did not influence ROS
   levels ([104]Figure 5). The reproductive clonogenic viability of AML12
   cells 24 hours after FFA treatment was determined using the clonogenic
   assay ([105]Figure 6). The dose that gave 5% mean clonogenic survival
   (DL95) was lower after FFA treatment (FFA: 5.27 Gy, Control: 6.12 Gy),
   indicating that FFAs sensitized cells to ionizing radiation. However,
   FFA-triggered sensitization was counteracted by the administration of
   miR-466e inhibitor, suggesting that miR-466e expression might influence
   radiation-sensitivity.

Figure 5. ROS levels in AML12 cells.

   Figure 5
   [106]Open in a new tab

   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.

   [107]Figure 6
   [108]Open in a new tab

   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.

Discussion

   Given the importance of diet in the development of obesity-associated
   metabolic syndromes, a significant amount of interest has been given to
   the molecular analysis of metabolic disruptions in obesity. In
   particular, there is growing evidence showing that diet can strongly
   influence epigenetic processes [109][21]. For this reason, we wondered
   whether metabolic disturbances associated with obesity were susceptible
   to influence radiation sensitivity.

   In order to understand the combinational effects of DIO and ionizing
   radiation in mice, we exposed C57BL/6J and C3H mice to fractionated
   doses of X-rays. DIO mice exhibit excessive body weight, body fat
   accumulation, prediabetes type 2 and metabolic syndrome. C3H mice were
   also studied because they are comparatively resistant to metabolic
   syndrome and atherosclerosis [110][54]. High 60% fat (HF) chow provided
   an energy density of 5.24 kcal/g (compared to 3.85 kcal/g for the
   control 10% fat chow) and mice fed a HF diet exhibited an accumulation
   of body fat and became obese, while mice fed a control diet did not.

   There are ample amounts of evidence describing increased bone marrow MN
   frequency after radiation exposure in mice [111][55]–[112][59]. On the
   contrary, HF diet did not modulate bone marrow MN frequency both in
   non-irradiated and irradiated mice, in contradiction with human data
   describing a correlation between peripheral blood MN frequency and
   pathologies associated with metabolic syndrome and obesity [113][60].
   The P/N ratio was higher in C3H than in C57BL/6J control mice,
   suggesting that these mice have a higher rate of erythropoiesis.
   Whereas no significant proliferation changes were observed in C57BL/6J
   mice, both exposure to radiation and HF diet resulted in lower
   erythroid precursors cell proliferation in C3H mice. In obese animals,
   it was suggested that bone marrow adipogenesis may impair
   erythropoiesis, resulting in anemia [114][61]. Rapidly cycling early
   erythroid progenitors are highly radiosensitive and encounter cell
   cycle damage checkpoints and apoptosis in response to radiation,
   compared to more mature erythroid cells [115][62]. Interestingly, this
   inhibition of proliferation was not observed in obese C3H mice after
   irradiation, suggesting that the progenitor cells might not respond to
   radiation exposure the same way in obese mice than in control mice, or
   that in response to both HF diet and radiation exposure, erythropoiesis
   might be stimulated. Additional investigations are necessary to
   understand the underlying mechanisms.

   Obesity and obesity-related complications play a significant role in
   the pathogenesis of liver diseases and liver cancer [116][63],
   [117][64]; for this reason, we focused our analysis on the interplay
   between radiation-responses and DIO in the mouse liver. Because DNA
   methylation is known to play a crucial role in carcinogenesis by
   regulating the expression of tumor suppressor or DNA repair genes
   [118][65], we measured the methylation profiles of several genes
   associated with liver cancer. While short-term modulation of promoter
   methylation was previously observed in C57BL/6J mice exposed to acute
   or chronic X-ray irradiation [119][66], only the Mgmt gene presented a
   methylated form after irradiation in animals fed a normal diet. Mgmt
   plays a major role in protecting cells from the genotoxic and
   carcinogenic effects of methylating mutagens [120][67]. Increased
   methylation of the Mgmt gene promoter might result in lower Mgmt gene
   expression levels, in contradiction with previous observations showing
   that genotoxic stress (including ionizing radiation) can induce Mgmt
   expression in rat liver cells [121][68], [122][69]. However, MSP does
   only provide qualitative results at best and additional investigations
   are required to measure Mgmt gene expression levels in our model. Our
   results also suggested that DIO was associated with increased
   hypermethylation of Ep300, p16 and Dapk gene promoters. Inactivation of
   tumor suppressor p16 by aberrant methylation is frequently observed in
   carcinomas and precancerous lesions of various organs [123][48],
   [124][49]. Similarly, aberrant methylation of the apoptosis-related
   Dapk promoter is extremely common in cancers [125][34], [126][46].
   These tumor suppressor genes exhibited promoter hypermethylation only
   in obese animals, suggesting potential effects in terms of radiation
   response and carcinogenesis. Expression levels of p16, Mgmt, Dapk,
   Ep300 and Socs1 were systematically lower in obese and irradiated
   animals, suggesting that promoter methylation contributes to gene
   repression in these animals.

   In a similar model of DIO in C57BL/6J mice, microarray analysis
   revealed that the expression of 97 genes was significantly modulated in
   the livers of mice fed a HF diet, compared to mice fed a normal-fat
   diet [127][70]. These genes were mainly involved in metabolism, stress
   defense mechanisms (such as protection against oxidative damage) and
   inflammatory processes. Other microarray analyses revealed that a
   number of genes are deregulated in response to ionizing radiation
   [128][71], [129][72], including at very low dose-rates [130][73],
   [131][74]. Because gene expression can be modulated by miRNAs, we
   studied diet- and radiation-associated miRNA regulations in C57BL/6J
   mouse livers.

   Recent studies have described obesity-related miRNA regulations in
   mouse and human models. These miRNAs were involved in various processes
   such as adipocyte differentiation, metabolic integration, insulin
   resistance and appetite regulation [132][75]. HF diet-associated miRNA
   up- (miR-22, miR-342-3p, miR-142-3p and others) and down-regulations
   (miR-200b, miR-200c, miR-204 and others) were observed in the adipose
   tissues of C57BL/6J mice [133][76]. In the liver, there is convincing
   evidence that miR-122 is involved in cholesterol and lipid metabolism,
   and the silencing of miR-122 has resulted in decreased cholesterol
   levels in mice and monkeys [134][77]–[135][79]. We did not observe any
   significant modulation of miR-122 in the livers of obese and/or
   irradiated mice. However, using SAM analysis, we identified a new HF
   diet-associated miRNA upregulation (miR-466e); additionally, the
   modulation of miR-709 expression was under the fold-change cut-off
   value of 1.5 but was potentially associated with HF diet as suggested
   by SAM analysis. miR-709 was recently described as a tumor-suppressor
   miRNA targeting Myc, Akt and Ras, which participates in the regulation
   of apoptosis through the miR-15a/miR-16-1 pathway [136][80], [137][81].
   HF diet resulted in the down-regulation of three putative miR-466e
   target genes (Zfp704, Cnot7, Cplx2), and it is highly likely that a
   number of genes are regulated in response to miR-466e up-regulation. A
   functional analysis of the putative target genes of miR-709 and
   miR-466e suggested that these miRNAs might be involved in lipid
   metabolism. miR-466e gene is included in the Chromosome 2 miRNA cluster
   (C2MC, or Sfmbt2 cluster), a mouse-specific large miRNA cluster located
   in intron 10 of the Sfmbt2 gene. C2MC contains 71 closely related miRNA
   genes modulated in response to cellular stress (nutrition deprivation,
   hyperglycemia, hypertonic stress, oxidative stress, xenobiotic stress,
   aging…) and involved in cell proliferation and apoptosis, cell fate
   decision and immune response [138][82]–[139][85].

   For this reason, we hypothesized that miR-466e might also be involved
   in stress response in the mouse liver, and we evaluated in vitro the
   effects of free fatty acid (FFA) administration and the potential role
   of miR-466e in the response of AML12 mouse liver cells to radiation.
   Although miR-466e inhibition did not modulate FFA-induced ROS levels,
   it counteracted the radiosensitization of AML12 cells by FFAs.
   Interestingly, it was recently shown that oxidative stress triggered
   acetylation in the miR-466h-5p promoter region, leading to the
   activation of this miRNA closely related to miR-466e [140][82]. Further
   investigations are needed to understand whether miR-466e is regulated
   through similar mechanisms, and to decipher the biological function of
   miR-466e.

   We observed that a number of miRNAs were upregulated after irradiation
   in mice fed a normal diet. A number of other studies have recently
   identified miRNAs associated with radiation responses [141][86],
   [142][87]. In particular, miR-21, which targets many genes in the
   apoptotic pathway, was consistently seen to be upregulated after
   exposure to ionizing radiation in various models [143][88]–[144][91];
   miR-21 was also upregulated in radiation-induced thymic lymphoma in
   BALB/c mice [145][92]. Many of the deregulated miRNAs in our study are
   not yet fully characterized and additional investigations will likely
   provide more insights into their potential involvement in radiation
   responses. Many of the molecular pathways identified by the miRPath
   tool in irradiated mice fed a normal diet were known to be associated
   with radiation responses and/or cancer, such as the TGF-β [146][93],
   MAPK [147][94], ErbB [148][95] and Wnt [149][53] signaling pathways,
   apoptosis, etc. Interestingly, these radiation-induced miRNA
   regulations were not observed in irradiated obese mice. This could not
   be explained by a previous up-regulation associated with HF diet, since
   only one miRNA was deregulated in obese mice.

   We therefore suggest that the molecular pathways normally involved in
   radiation responses are disrupted in obese mice. The question remains
   whether those might influence long-term effects, such as
   radiation-induced carcinogenesis. It is highly possible that because
   obese animals experience chronic oxidative stress (as reported by
   others and suggested by our immunohistochemistry results) [150][96],
   they are further sensitized to ionizing radiation risks. 8-OHdGs in CpG
   sequences were shown to inhibit the methylation of neighboring cytosine
   residues, resulting in DNA hypomethylation [151][97]. However, obesity
   was associated in our model with increased promoter methylation for
   several genes.

   A substantial body of evidence shows that oxidative stress is a major
   contributor to the deleterious effects of obesity [152][98]. The
   expansion of abdominal fat results in elevated levels of free fatty
   acids (FAAs) [153][99], [154][100], which stimulate reactive oxygen
   species (ROS) production [155][98] and induce hepatic insulin
   resistance as well as increased glucose production. Furthermore,
   decreased antioxidant capacity levels and increased DNA damage were
   described in relationship with obesity-induced chronic oxidative stress
   [156][96]. The disruption of genetic and epigenetic mechanisms by
   obesity-induced chronic oxidative stress might result in a deficient
   response to ionizing radiation. For example, it was shown that the
   deregulation of protein kinase C (PKC) signaling, an early responder to
   ionizing radiation [157][101], is a crucial event in obesity-related
   metabolic syndrome and oxidative stress [158][102], [159][103].
   Irradiation in obese animals might thus result in increased cancer
   risk. Furthermore, miRNA regulations (such as the upregulation of
   miR-466e) are likely to play a significant role in regulating radiation
   response.

   Overall, it is necessary to better understand the crosstalk between
   obesity-related metabolic syndrome and radiation responses, both at low
   and high dose ranges, in order to better evaluate the risks and effects
   of ionizing radiation resulting from natural or medical exposures, or
   to develop specific radiation-therapy regimens adapted to each patient
   characteristics.

Supporting Information

   Figure S1

   Time-course of weight gain in mice. C57BL/6J (left) and C3H (right)
   mice were fed a normal (black symbols) and a HF diet (colored symbols).

   (PDF)
   [160]Click here for additional data file.^ (122.1KB, pdf)
   Figure S2

   Heatmap of miRNA expression in the livers of obese and/or irradiated
   C57BL/6J DIO mice. Unsupervised hierarchical clustering of 1157 mouse
   miRNAs (100% of known mouse miRNAs) was performed. C: control (mice No.
   116, 117, 118); HF: HF diet (mice No. 106, 107, 108); R: irradiated
   (mice No. 111, 112, 113); HFR: HF diet, irradiated (mice No. 101, 102,
   103).

   (PDF)
   [161]Click here for additional data file.^ (76.4KB, pdf)
   Table S1

   Real-time PCR verification of miRNA expression. Expression ratios
   (compared to control) of miR-466e-3p, miR-185-3p and miR-21-3p were
   measured by RT-PCR. Green values indicate miRNAs identified as
   upregulated in the SAM microarray analysis.

   (PDF)
   [162]Click here for additional data file.^ (42.4KB, pdf)

Acknowledgments