A transgenerational carcinogenic effect of chromium(III) has been demonstrated in mice, in which treatment to male mice increases tumor risk in their offspring. In search of a mechanism for this effect, we carried out differential DNA methylation analysis of sperm DNA. We identified allele-specific hypomethylation of the upstream spacer-promoter region of the 45S ribosomal RNA gene after chromium(III) exposure. To determine if this epigenetic change can be transmitted to the offspring, day-8 mouse embryos from treated and control groups were examined for the level of DNA methylation and allele frequency in the 45S ribosomal RNA promoter. Bisulfite/cloning/sequencing assay detected an overall lower level of methylation at 27 45S ribosomal RNA CpG sites in the treated (10 embryos from 3 families) than in the control (10 embryos from 5 families) group (paired-T test, p<0.0001). For fast allele typing and methylation quantification from the entire gene pool, rather than from selected clones, the pyrosequencing technique was used. This revealed that the frequency for T-allele varied among families (2 from treated and 5 from control group). Also, DNA methylation was inversely correlated with the frequency of T-allele regardless of chromium(III) treatment. Detection of hypomethylation in the offspring provides the first evidence of epigenetic inheritance of a gene that is not classified as imprinted. Implications of this finding, once confirmed in a large sample, for transgenerational carcinogenesis are exciting; ribosomal RNA has a major role in growth control and putatively in cancer. The inverse relationship of allele frequency and DNA methylation in the 45S ribosomal RNA indicates an interesting fine control over the methylation process. Methylation needs to be adjusted for allele frequency for this gene.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]