Endogenous methanol regulates mammalian gene activity

Abstract

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.

Figure 1. Dynamics of methanol, formaldehyde and ethanol changes in the blood plasma of mice after 4-MP administration.

Each mouse in the treatment and control groups received an intraperitoneal injection of 4-MP (10 mg/kg) or the saline solution, respectively. Blood samples were analyzed for methanol/ethanol and formaldehyde content by GC and HPLC analyses, respectively. The data are shown with standard error bars, and P-values (Student's t-test) are designated by: ***, P<0.001; *, P<0.05; n.s., not significant.

Figure 2. Microarray analysis of differentially regulated murine brain mRNAs after 4-MP or methanol administration.

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 P-value <0.05.

Figure 3. Diagram of biological processes with differentially expressed genes after 4-MP and methanol administration.

Genes significantly differentially expressed in treated and control mice were analyzed using the Gene Ontology (GO) tool in the PANTHER database to categorize the biological processes in which they participate.

Figure 4. Verification of microarray data with qRT-PCR.

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

Figure 5. qRT-PCR analysis of murine brain mRNAs content after intraperitoneal 4-MP administration.

The data shown represent five independent experiments. ***, P<0.001 (Student's t-test).

Figure 6. The examination of the putative role of intestinal microbes in the generation of methanol in rats.

A - Scheme of the rat gastrointestinal tract resection. 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 P-values (Student's t-test) are indicated.

Figure 7. Rat liver ADH suppression results in an increase of endogenous methanol and ethanol.

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 P-values (Student's t-test) are indicated.

Figure 8. The suppression of mouse liver ADH results in the accumulation of mRNA of the ADH/AlDH gene cluster.

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