Dietary Methanol Regulates Human Gene Activity

<div><p>Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, <i>HBA</i> and <i>HBB</i>, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, <i>GAPDH</i> and <i>SNX27</i>, and genes revealed in this study, including <i>MME</i>, <i>SORL1</i>, <i>DDIT4</i>, <i>HBA</i> and <i>HBB</i>. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.</p></div

List of significantly down- and up-regulated genes in the WBCs of volunteers after citrus pectin intake.

<p>*KEGG, Kyoto Encyclopedia of Genes and Genomes (<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a>).</p><p>**The National Center for Biotechnology Information (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>).</p>&<p>Genes selected for further analysis.</p

Endogenous Methanol Regulates Mammalian Gene Activity

<div><p>We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.</p></div

Microarray analysis of differentially regulated murine brain mRNAs after 4-MP or methanol administration.

<p>Venn diagram of the genes that are differentially expressed after 4-MP and methanol administration compared to the control mice after saline solution injection. Genes were analyzed using the J-Express gene expression analysis software. The number of genes commonly regulated is indicated by the intersection of the circles. All the genes included in this analysis had significant changes in their expression compared to the control, with a <i>P</i>-value <0.05.</p

qRT-PCR analysis of WBC mRNA content after red wine intake.

<p>The relative mRNA quantities after red wine intake was normalized to the mRNA levels before red wine intake. Student’s <i>t</i>-test <i>P</i>-values were calculated by using triple bloods samples of three volunteers.</p

Rat liver ADH suppression results in an increase of endogenous methanol and ethanol.

<p>Measurements of liver methanol and ethanol content 30-MP (10 mg/kg) administration directly into the portal vein. The data are shown with standard error bars, and <i>P</i>-values (Student's <i>t</i>-test) are indicated.</p

Microarray analysis of differentially regulated murine brain mRNAs after mouse inhalation of methanol and wounded leaf vapors.

<p>(A) Experimental set-up for the inhalation of vapors from wounded leaves by the mice. (B) Venn diagram of the genes that are differentially expressed after the inhalation of methanol and wounded leaf vapors compared with those after the inhalation of water vapor. The genes were analyzed using the J-Express gene expression analysis software. The number of genes commonly regulated is indicated in the intersection of the circles. All the genes (with an average fold-change ≥1.3-fold) included in this analysis showed significant changes in their expression compared with the control, with a <i>Q</i>-value <0.05.</p

The suppression of mouse liver ADH results in the accumulation of mRNA of the ADH/AlDH gene cluster.

<p>The liver mRNA was quantified by qRT-PCR after 4-MP (10 mg/kg) administration. The data are shown with standard error bars, and <i>P</i>-values (Student's <i>t</i>-test) are indicated.</p

Verification of microarray data with qRT-PCR.

<p>Murine brain mRNAs were quantified by qRT-PCR after treatment with methanol by inhalation. The data shown represent five independent experiments. ***, <i>P</i><0.001 (Student's <i>t</i>-test).</p

The examination of the putative role of intestinal microbes in the generation of methanol in rats.

<p><b>A</b> - Scheme of the rat gastrointestinal tract resection. <b>B</b> - The diagram showing levels of methanol in the blood of rats before and after resection of the gastrointestinal tract. The data are shown with standard error bars, and <i>P</i>-values (Student's <i>t</i>-test) are indicated.</p