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Ethanol-induced upregulation of 10-formyltetrahydrofolate dehydrogenase helps relieve ethanol-induced oxidative stress.

ABSTRACT: Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the -124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBP? was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBP? helps relieve embryonic oxidative stress induced by ethanol exposure.

SUBMITTER: Hsiao TH

PROVIDER: S-EPMC3911513 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Ethanol-induced upregulation of 10-formyltetrahydrofolate dehydrogenase helps relieve ethanol-induced oxidative stress.

Hsiao Tsun-Hsien TH Lin Chia-Jen CJ Chung Yi-Shao YS Lee Gang-Hui GH Kao Tseng-Ting TT Chang Wen-Ni WN Chen Bing-Hung BH Hung Jan-Jong JJ Fu Tzu-Fun TF

Molecular and cellular biology 20131125 3

Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approx ...[more]

PMID: 24277932

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Project description:Folate (vitamin B9) and its biologically active derivatives are well-known antioxidant molecules protecting cells from oxidative degradation. The presence of high glucose, often found in diabetic patients, causes oxidative stress resulting in cellular stress and inflammatory injury. Cells in organs such as the lung are highly prone to inflammation, and various protective mechanisms exist to prevent the progressive disorders arising from inflammation. In the present study, the synthetic form of folate, i.e. folic acid, and active forms of folate, i.e. 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, were evaluated for their antioxidant and antiinflammatory potential against high glucose (50 mM)-mediated oxidative stress and inflammation in BEAS-2B cells, an immortalised bronchial epithelial cell line. High glucose treatment showed a 67% reduction in the viability of BEAS-2B cells, which was restored to the viability levels seen in control cultures by the addition of active folate derivatives to the culture media. The DCFH-DA fluorometric assay was performed for oxidative stress detection. The high glucose-treated cells showed a significantly higher fluorescence intensity (1.81- and 3.8-fold for microplate assay and microscopic observation, respectively), which was normalised to control levels on supplementation with active folate derivatives. The proinflammatory NF-κB p50 protein expression in the active folate derivative-supplemented high glucose-treated cells was significantly lower compared to the folic acid treatment. In support of these findings, in silico microarray GENVESTIGATOR database analysis showed that in bronchiolar small airway epithelial cells exposed to inflammatory condition, folate utilization pathway genes are largely downregulated. However, the folate-binding protein gene, which encodes to the folate receptor 1 (FOLR1), is significantly upregulated, suggesting a high demand for folate by these cells in inflammatory situations. Supplementation of the active folate derivatives 5-methyltetrahydrofolate and 10-formyltetrahydrofolate resulted in significantly higher protection over the folic acid from high glucose-induced oxidative stress and inflammation. Therefore, the biologically active folate derivatives could be a suitable alternative over the folic acid for alleviating inflammatory injury-causing oxidative stress.

Project description:During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

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Ethanol-induced upregulation of 10-formyltetrahydrofolate dehydrogenase helps relieve ethanol-induced oxidative stress.

Publications

Ethanol-induced upregulation of 10-formyltetrahydrofolate dehydrogenase helps relieve ethanol-induced oxidative stress.

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