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Dietary heme mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

ABSTRACT: Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.

ORGANISM(S): Mus musculus

PROVIDER: GSE37006 | GEO | 2012/08/22

SECONDARY ACCESSION(S): PRJNA157869

REPOSITORIES: GEO

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Project description:Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARM-NM-1 target genes. The aim of this study was to investigate the role of PPARM-NM-1 in heme-induced hyperproliferation and hyperplasia. Male PPARM-NM-1 KO and WT mice received a purified diet with or without heme. As PPARM-NM-1 is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARM-NM-1 leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARM-NM-1 in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARM-NM-1 KO mice. We conclude that PPARM-NM-1 plays a protective role in colon against oxidative stress, but PPARM-NM-1 does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia. Wild type and peroxisome proliferator-activated receptor alpha (PPARM-NM-1) knockout mice were fed a Westernized high fat diet, or the same diet supplemented with 0.5 M-BM-5mol heme/g diet. After 14 days of intervention, mice were killed and gene expression was profiled in colon.

Project description:Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signalling as recently described. It is unknown whether the change in signaling is caused by cytotoxic stress and/or by oxidative stress, as these processes were never studied separately. Therefore, the aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified humanized control diet or this diet supplemented with 0.2 Âµmol heme/g. Oxidative stress was measured as Thiobarbituric Acid Reactive Substances (TBARS) in fecal water. Cytotoxicity of fecal water was quantified with a bioassay. Epithelial cell proliferation was determined by Ki67 immunohistochemistry and mucosal responses were further studied in detail by whole genome transcriptomics. Dietary heme caused instantaneous and delayed changes in the luminal contents which were reflected in the mucosa. Instantaneous, there was an increase in reactive oxygen species leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an instantaneous antioxidant response and PPAR target gene activation. After day 4 cytotoxicity of the colonic contents was increased and hyperproliferation was initiated, indicating that cytotoxicity was causal for the initiation of hyperproliferation. Several oncogenes were activated and tumor protein 53 was inhibited. In conclusion, dietary heme caused an instantaneous production of reactive oxygen species in mouse colon. A lag time was observed in the formation of cytotoxicity which coincided with the initiation hyperproliferation. Keywords: expression profiling by array Mice were fed a Westernized high fat control diet, or the same diet supplemented with 0.2 Âµmol heme/g diet. After different days of intervention, mice were killed and gene expression was profiled in colon.

Project description:Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signalling as recently described. It is unknown whether the change in signaling is caused by cytotoxic stress and/or by oxidative stress, as these processes were never studied separately. Therefore, the aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified humanized control diet or this diet supplemented with 0.2 µmol heme/g. Oxidative stress was measured as Thiobarbituric Acid Reactive Substances (TBARS) in fecal water. Cytotoxicity of fecal water was quantified with a bioassay. Epithelial cell proliferation was determined by Ki67 immunohistochemistry and mucosal responses were further studied in detail by whole genome transcriptomics. Dietary heme caused instantaneous and delayed changes in the luminal contents which were reflected in the mucosa. Instantaneous, there was an increase in reactive oxygen species leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an instantaneous antioxidant response and PPAR target gene activation. After day 4 cytotoxicity of the colonic contents was increased and hyperproliferation was initiated, indicating that cytotoxicity was causal for the initiation of hyperproliferation. Several oncogenes were activated and tumor protein 53 was inhibited. In conclusion, dietary heme caused an instantaneous production of reactive oxygen species in mouse colon. A lag time was observed in the formation of cytotoxicity which coincided with the initiation hyperproliferation. Keywords: expression profiling by array

Project description:Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function. Mice were fed a Westernized high fat control diet, or the same diet supplemented with 0.5 µmol heme/g diet. One group of control and one group of heme mice received a mixture of broad spectrum Antibiotics (Abx) (ampicilin, neomycin and metronidazole) in their drinking water. After 14 days of intervention, mice were killed and gene expression was profiled in colon.

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Dietary heme mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

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