Discussion
There is increasing evidence suggesting the early life environment has an impact on multigenerational health. This study characterizes the intergenerational effects of iAs on offspring health, as inherited through the female germline. We provide evidence that iAs has sex-specific effects, such that dysregulated glucose metabolism affects both F1/F2 females and F2 males. More specifically, these effects were dose-specific with a late life onset. We show exposed F1 and F2 males have impaired growth as indicated by weekly body weight, where only a subset of the exposed F2 female lineage had surpassed the weight gain of F2 female control mice. We probed DNA methylation changes as a potential mechanism for intergenerational damage. We found DMCs and DMRs occur across sex and generations, identifying genes with high DMC/DMR content. Together, this data suggests in utero arsenic exposure alters metabolic phenotypes into adulthood of despite the absence of iAs exposure during adult lifetimes though with limited recapitulation of methylation changes across generations. The effects presented are sex-, dose-, and generation-specific indicating the prenatal environment influences the onset of disease that persists throughout generations; more studies exploring the intergenerational effects on epigenetic inheritance and the associated functionality are needed to link iAs exposure to changes in gene expression and protein function.
It has been well established that maternal in utero iAs exposure is associated with dysregulated glucose metabolism in the F1 populations(Young, Cai, and States 2018; Navas-Acien et al. 2019; Tinkelman et al. 2020). In a rat model, the maternal exposure of 500 ppb and 50 ppm throughout gestation and 2 months post-partum resulted in the onset of T2D phenotype in F1 offspring(Bonaventura et al. 2017). Mouse studies investigating in utero iAs of 100 ppb also identify diabetes related phenotypes such as increased plasma insulin, decreased pancreatic insulin production, or the onset of non-alcoholic fatty liver disease within offspring(Huang et al. 2018; Martin, Stýblo, and Fry 2017; Ditzel et al. 2016). Unlike our study using standard chow, these studies are coupled with nutritional arms (total western diet or supplementation with folate) and doses of iAs that exceed doses found in drinking water. Our exposure paradigm was designed to identify if iAs targeted the developing fetus and germ cells using human relevant exposures. Thus, our results indicate the dysregulated glucose metabolism phenotype in F1 and F2 generations are present at doses relevant to the human population.
Our findings on generational impacts on body weight and fat mass contributes to the growing body of in utero iAs exposure on offspring metabolism. Our data show maternal exposure causes sex- and dose-specific weight changes in F1 offspring. Additionally, grand maternal exposure alters the life-long body composition in both male and female F2 offspring. Previous studies using exposures ranging from 10 ppb – 42.5 ppm show maternal iAs exposure contributes to higher body weight in female offspring(Rodriguez et al. 2016a). Additionally, a study using a similar exposure of 250 ppb found no changes in body weight within the F1 offspring, early life stunted growth in F2 offspring that was restored in late life, and increased adiposity in F3 males(Gong et al. 2021). These studies focusing on rodent models are but a few indicating dose of iAs and sex have a role in adiposity. Further research is needed to identify the influence of iAs early- and late-life body composition and the implications on human metabolic health.
Our data show glucose metabolism and body composition are dysregulated by in utero iAs exposure, yet it is unclear if the onset of disease in our study is influenced by epigenetic alterations or other factors. The onset of T2D in adults chronically exposed to iAs may be a result of non-epigenetic mechanisms such as inhibition of a GLUT4, damage to ß-cells by ROS production, inhibition of glucose simulated insulin secretion, or increased stimulation of liver gluconeogenesis(Martin, Stýblo, and Fry 2017). Chronic iAs exposure is known to cause increased body weight and alterations in lipid metabolism(Castriota et al. 2018; C et al. 2019). Additionally, studies show the maternal metabolic milieu influences the onset of obesity in offspring ranging from adolescence and into adulthood(Tequeanes et al. 2009; Derraik et al. 2015). Despite the role of iAs in direct damage to tissues and the maternal metabolism, increasing evidence from mouse and human studies shows developmental iAs exposure alters DNA methylation in offspring with associations to metabolic phenotypes. In a longitudinal mother-child cohort in Bangladesh, differential methylation of CpG sites within in blood mononuclear cells were characterized, where 12 hypermethylated CpG sites were associated with prenatal iAs exposure(Gliga et al. 2018). Of the differentially methylated CpG sites, several were associated with proteins essential to insulin secretion by ß-cells in the pancreas. Similar to previously reported studies, our data finds differential methylation within genes associated with T2D(Ali 2013) and obesity(Loos and Yeo 2021) - Pik3c2g, Ptprn2 ,Adcy5, Slc27a4, Irs2 , Tcf12, Jazf1, Adcy5, Slc27a, Adipor1 , Fto , Pparg, and Adcy3 . While in utero exposure is known to cause long-term health effects in developing fetuses and young children, often impacting the establishment of DNA methylation(Smeester and Fry 2018), further research is needed to explore the association between our DMCs and the onset of disease. It is clear the identification of differential methylation in offspring is associated with maternal iAs exposure, where differential methylation may be a plausible contributor to the onset of metabolic phenotypes.
Sex-, dose-, and generation-specific effects are common among prenatal environmental exposures. Previous studies have reported similar effects of paternal iAs exposure on glucose homeostasis through the F1 and F2 generations(Gong et al. 2021). Glucose intolerance and hepatic insulin resent was present in F1 females, not F1 males, without altering body weight. Despite low body wean weights, body weight was restored into adulthood within the F2 generation. In contrast to our study, Gong et. al explores the transmission of metabolic phenotypes through the paternal lineage (mating F1 male offspring), not the maternal lineage (mating F1 female offspring). These results indicate a paternal epigenetic influence on adult F1 and F2 offspring metabolic disease phenotypes. Another study exploring both maternal and paternal exposure on transgenerational health found altered DNA methylation and reproductive phenotypes(Nava-Rivera et al. 2021). Methylation in the ovary and testes was significantly lower in the F1 generation, remained unchanged in the F2 generation, and increased within the F3 generation. Sperm quality parameters were also lower in the F1 and F3 male offspring, not within the F2 offspring. Results from these two intergenerational studies are foundational for the understanding of iAs exposure and epigenetic reprogramming. Our shared DMC containing genes across sex and generation provides an exciting foundation for genes targeted by prenatal iAs exposure. Despite the absence of overlap between generations, it is possible CpG methylation within the detected DMCs was re-established during F2 embryogenesis. Our findings could indicate female PGCs may retain epigenetic damage from F0 exposure that persists regardless of epigenetic reprogramming. Our results join the few novel studies indicating intergenerational iAs exposure alters metabolic phenotypes and the epigenome in a sex-, dose-, and generation-specific manner.