Altered DNA Methylation Induced In Utero May Cause Multigenerational Abnormal Development: Role of Epigenetic Inheritance

Francisco Cisneros
Preclinical Services; Charles River Laboratories

It was believed that DNA methylation was cleared and reset in the germ line and/or during embryogenesis. In recent years, evidence indicates that epigenetic inheritance exists and possibly represents a major source of phenotypic variation and sporadic disease. Effects of certain environmental pollutants in DNA methylation have been reported. The role of DNA methylation in embryo development is well documented. The objective was to determine whether an intrauterine 5-AZA-2'-deoxycytidineine (5-AZA-CdR), a demethylating agent, exposure would induce transmissible alterations in development, if such insult causes heritable global DNA methylation changes correlating with the observed alterations, and whether the treatment alters methylation levels of promoter regions of specific developmental related genes. Pregnant mice (F0) were injected intra-peritoneally (i.p.) with 1 mg/kg of 5-AZA-CdR at gestation day (GD) 10. Male and female offspring were later mated avoiding consanguinity. Two generations were examined. In each generation, 50% of pregnant mice were killed and fetuses examined via skeletal analysis and necropsy. Global DNA methylation levels were determined using the cytosine extension assay. The quantitative HpaII-PCR assay was used to analyze methylation status of gene promoter regions. Leg and tail abnormalities, retarded postnatal development, cleft palate and abnormal male mating behavior as well as global DNA hypermethylation levels (in palate tissues of GD 17 fetuses) were observed in 5-AZA-CdR F1 offspring. In F2, cleft palate and global DNA hypermethylation levels were evident. This data suggest that an intrauterine insult from 5-AZA-CdR exposure induces alterations of methylation patterns which may be inherited by the offspring of exposed parents causing altered development.