14C methanol incorporation into DNA and proteins of organogenesis stage mouse embryos in vitro

Methanol (MeOH), a widely used industrial solvent and alternative motor fuel, has been shown to be mutagenic and teratogenic. We have demonstrated that methanol is teratogenic in mice in vivo and causes dysmorphogenesis in cultured organogenesis stage mouse embryos. Although MeOH is a product of endogenous metabolism in the gut and can be found in humans following consumption of various foods, elevated levels of methanol could lead to methylation of cellular macromolecules. DNA methylation has been demonstrated to suppress transcription of fetal genes and may also play an important role in genetic imprinting. Embryonal proteins are also potential targets for methanol-induced methylation. We investigated the potential of administered methanol to incorporate into and/or alter the methylation of embryonal DNA or to affect specific protein methylation. Gestational day 8 CD-1 mouse embryos were grown for 24 h in culture medium (CM) with 0, 4, or 8 mg MeOH 1 20mCi 14 C-MeOH/mL. At the end of the culture period, yolk sacs and embryos were separated for each treatment group. The DNA was purified by cesium chloride gradient centrifugation in the presence of ethidium bromide and 14 C incorporation was determined. Methylation of a selected gene, Hoxc-8, was assessed by using methylation-specific restriction enzymes. The 14 C activity was found superimposed over the DNA-containing fraction, indicating incorporation. DNA from embryos treated with 4 mg MeOH/mL CM gave the highest incorporation of 14 C-MeOH (8 mg/mL was growth inhibiting). Methylation of Hoxc-8 appeared to be increased in embryos treated with 4 mg MeOH/mL CM, but not in embryos treated with 8 mg MeOH/mL. Lack of incorporation of methylation at the higher concentration may be due to the failure of embryos to grow at this concentration of MeOH. The incorporation of 14 C-MeOH into embryo proteins was investigated by polyacrylamide gel electrophoresis (PAGE) and autoradiography. Incorporation of 14 C-MeOH into specific proteins was observed but the labeling specificity was not methanol dose-related. These results indicate that methyl groups from 14 C-MeOH are incorporated into mouse embryo DNA and protein. Our results further suggest that methanol exposure may increase genomic methylation under certain conditions which could lead to altered gene expression. © 2001 Elsevier Science Inc. All rights reserved.