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

Methanol (MeOH), a widely used industrial solvent and alternative motor fue l, has been shown to be mutagenic and teratogenic. We have demonstrated tha t methanol is teratogenic in mice in vivo and causes dysmorphogenesis in cu ltured organogenesis stage mouse embryos. Although MeOH is a product of end ogenous metabolism in the gut and can be found in humans following consumpt ion of various foods, elevated levels of methanol could lead to methylation of cellular macromolecules. DNA methylation has been demonstrated to suppr ess transcription of fetal genes and may also play an important role in gen etic imprinting. Embryonal proteins are also potential targets for methanol - induced methylation. We investigated the potential of administered methan ol to incorporate into and/or alter the methylation of embryonal DNA or to affect specific protein methylation. Gestational day 8 CD-1 mouse embryos w ere grown for 24 h in culture medium (CM) with 0, 4, or 8 mg MeOH + 20 mu C i C-14-MeOH/mL. At the end of the culture period, yolk sacs and embryos wer e separated for each treatment group. The DNA was purified by cesium chlori de gradient centrifugation in the presence of ethidium bromide and C-14 inc orporation was determined. Methylation of a selected gene, Hoxc-8, was asse ssed by using methylation-specific restriction enzymes. The C-14 activity w as found superimposed over the DNA-containing fraction, indicating incorpor ation. DNA from embryos treated with 4 mg MeOH/mL CM gave the highest incor poration of C-14-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 a t this concentration of MeOH. The incorporation of C-14-MeOH into embryo pr oteins was investigated by polyacrylamide gel electrophoresis (PAGE) and au toradiography. Incorporation of C-14-MeOH into specific proteins was observ ed but the labeling specificity was not methanol dose-related. These result s indicate that methyl groups from C-14-MeOH are incorporated into mouse em bryo DNA and protein. Our results further suggest that methanol exposure ma y increase genomic methylation under certain conditions which could lead to altered gene expression. (C) 2001 Elsevier Science Inc. All rights reserve d.