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.