3.6 Preferential Hypermethylation in DMCs and DMRs of F1 Generation
To identify a potential mechanism for epigenetic damage, we assessed differentially methylated CpGs (DMCs) and differentially methylated regions (DMRs) in the liver of 10 ppb and 245 ppb F1 mice (Table 1) since these dose levels are most relevant to human exposures. In the F1 animals of both sexes, we found an excess of hypermethylated CpG sites at both doses. Specifically, in F1 females, we found 259 (66%) hypermethylated and 132 (34%) hypomethylated DMCs within the 10-ppb group, and 157 (61%) hypermethylated and 99 (39%) hypomethylated DMCs within the 245-ppb group. Only 4 (80%) DMRs were hypermethylated and 1 DMR hypomethylated in the 10-ppb group, whereas no DMRs were identified in the 245-ppb group. In F1 males, we identified 299 (65%) hypermethylated and 196 (42%) hypomethylated DMCs within the 10-ppb group, and 265 (64%) hypermethylated and 151 (36%) hypomethylated DMCS in the 245-ppb group. Only 3 (43%) hypermethylated and 4 (57%) hypomethylated DMRs were identified in the 10 ppb iAs group, whereas 9 (82%) hypermethylated and 2 (18%) hypomethylated DMRs were identified in the 245-ppb group. Our results show F1 males have more dysregulated methylation in DMCs and DMRs compared to F1 females, regardless of the exposure group.
The F2 mice sustained similar overall numbers of differentially methylated CpG sites and recapitulated the hypermethylation bias seen in F1 animals. Our F2 results indicate higher DMC and DMR content within F2 females compared to F2 males, a contrast to the F1 generation (Table 1) . Females of the 10-ppb group had preferential DMC hypermethylation in the liver, where 320 (60%) hypermethylated and 217 (40%) hypomethylated DMCs were identified in the 10-ppb group. We identified 9 (56%) hypermethylated and 7 (44%) hypomethylated DMRs within the 10-ppb group. Females of the 245-ppb group had a slight bias towards hypomethylation, where 208 (57%) hypomethylated and 156 (43%) hypermethylated DMCs were identified. We found 1 (20%) hypermethylated and 4 (80%) hypomethylated DMRs in the 245 ppb F2 females. Within the 10-ppb F2 males, we identified 140 (54%) hypermethylated and 118 (46%) hypomethylated DMCs, detecting 1 hypermethylated DMR. F2 males in the 245-ppb group had 144 (52%) hypermethylated and 133 (48%) hypomethylated DMCs, and only 1 hypermethylated DMR. Our findings show female DMCs and DMRs within the liver are more reactive to prenatal exposure to iAs in both the 10 ppb and 245 ppb groups when compared to males.