Abstract
Inorganic Arsenic (iAs) is one of the largest toxic exposures to impact
humanity worldwide. Exposure to iAs during pregnancy may disrupt the
proper remodeling of the epigenome of F1 developing offspring and
potentially their F2 grand-offspring via disruption of fetal primordial
germ cells (PGCs). There is a limited understanding between the
correlation of disease phenotype and methylation profile within
offspring of both generations and whether it persists to adulthood. Our
study aims to understand the intergenerational effects of in
utero iAs exposure on the epigenetic profile and onset of disease
phenotypes within F1 and F2 adult offspring, despite the life-long
absence of direct arsenic exposure within these generations. We exposed
F0 female mice (C57BL6/J) to the following doses of iAs in drinking
water 2 weeks before pregnancy until the birth of the F1 offspring: 1
ppb, 10 ppb, 245 ppb, and 2300 ppb. We found sex- and dose-specific
changes in weight and body composition that persist from early time to
adulthood within both generations. Fasting blood glucose challenge
suggests iAs exposure causes dysregulation of glucose metabolism,
revealing generational, exposure, and sex specific differences. Toward
understanding the mechanism, genome-wide DNA methylation data highlights
exposure-specific patterns in liver, finding dysregulation within genes
associated with cancer, T2D, and obesity. We also identified regions
containing persistently differentially methylated CpG sites between F1
and F2 generations. Our results indicate F1 developing embryos and F2
PGCs retain epigenetic damage established during the prenatal period and
are associated with adult metabolic dysfunction.
Introduction
Arsenic (iAs) exposure via drinking water is one of the largest global
exposures affecting prenatal health, and has a growing body of evidence
that suggests gestational iAs exposure influences intergenerational
epigenetic inheritance(National Research Council (U.S.). Subcommittee on
Arsenic in Drinking Water. 1999; Hossain et al. 2017; Nohara et al.
2011; Nohara, Suzuki, and Okamura 2020). Data from mother-infant pairs
show prenatal iAs exposure alters DNA methylation(Xie et al. 2007; Zhao
et al. 2002; Tsang et al. 2012) and birth outcomes(Marie et al. 2018;
Laine et al. 2015; Gilbert-Diamond et al. 2016; Fei et al. 2013) in
newborns. Studies using rodent models find prenatal iAs exposure causes
type 2 diabetes (T2D)(Young, Cai, and States 2018; Liu et al. 2014),
obesity(Rodriguez et al. 2016b; C et al. 2019), and differential
methylation(Martin, Stýblo, and Fry 2017; Rojas et al. 2015; Bailey et
al. 2013) in adult mice. However, most rodent studies use doses of iAs
that are considered carcinogenic(“TOXICOLOGICAL PROFILE FOR ARSENIC
| Enhanced Reader,” n.d.) thus the role of methylation and
disease may be skewed, and methylation data reported from
epidemiological studies are confounded by co-exposures. Due to its
prevalence as a health threat in the United States and around the world,
it is important to characterize the multigenerational epigenetic damage
caused by iAs exposure and its long-term health effects in adult
offspring. However, there are no studies to date investigating the
intergenerational impact of maternal iAs exposure inherited through the
female germline.
Epigenetic inheritance occurs by escape from complete epigenetic
reprogramming. Epigenetic reprogramming is critical for regulation of
gene expression and tissue differentiation in the developing embryo, and
imperative for sex differentiation within primordial germ cells
(PGCs)(Feng, Jacobsen, and Reik 2010; Cedar and Bergman 2012).
Reprogramming occurs during early and late embryogenesis, where DNA
methylation is erased then re-established in two waves of
demethylation/re-methylation. The first reprogramming event takes place
within the primordial germ cells (F2, PGCs) of F1 offspring, and the
second in the somatic F1 post-fertilization pluripotent stem
cells(Sasaki and Matsui 2008). Since the epigenome is vulnerable during
development, disruption of epigenetic reprogramming by environmental
exposures impacts both the developing zygote (F1) and the PGCs (F2), and
is known to cause the onset of disease in subsequent generations(Painter
et al. 2008; Skinner and Guerrero-Bosagna 2014; Titus-Ernstoff et al.
2008). When the F1 PGCs develop into oocytes, become fertilized, and
undergo the waves of somatic demethylation/re-methylation as the
developing F2 zygote during pregnancy, it is unclear if grand-maternal
epigenetic damage caused by in utero iAs exposure will persist in
adult F2s, despite the reprogramming event. Thus, we designed an
exposure paradigm to 1) identify the intergenerational epigenetic
scarring caused by in utero iAs exposure and 2) assess the
contribution to the onset of disease in adulthood using a mouse model.
The epigenetic damage of iAs exposure during pregnancy is hypothesized
to be associated with iAs metabolism and the 1-carbon pathway. iAs
metabolism acts as an inhibitor of DNA methylation due to competition
for DNA methylation precursors. Biotransformation of iAs utilizes the
one carbon metabolism pathway, the same metabolic pathway that generates
methyl groups for the synthesis of DNA. The universal methyl donor,
S-aenosyl-l-methione (SAM), is utilized by iAs-3-methyltransferase
(AS3MT) to generate metabolites monomethylarsonic acid (MMA) and
dimethylarsonic acid (DMA) for urinary excretion(Spratlen et al. 2017).
SAM is also used by DNMTs to add methyl groups to convert unmodified
cytosines to 5-methylcytosine (5-mC)(Thomas, Waters, and Styblo 2004).
Since both AS3MT and DNMT1 compete for SAM, evidence suggests
utilization of SAM is responsible for the lower global methylation
changes seen in exposed individuals(Nohara et al. 2011; Zhao et al.
2002; Xie et al. 2007). Thus, in utero iAs exposure has the
potential to disrupt normal methylation patterning during epigenetic
reprogramming.
We hypothesized the disruption of DNA methylation by in utero iAs
exposure would target both the developing fetus (F1) and primordial germ
cell (F2) epigenomes leading to the onset of adulthood diseases. We
exposed F0 females to human relevant doses of iAs throughout pregnancy
that represented the WHO limit (10 ppb)(National Research Council
(U.S.). Subcommittee on Arsenic in Drinking Water. 1999), the highest
average global exposure (245 ppb) (Argos 2015), and the known prenatal
carcinogenic exposure in mice (2300 ppb)(Waalkes et al. 2004). Ourgestational exposure window was chosen to restrict the impact of
iAs to development and causally link early exposure to later-in-life
metabolic diseases. We assess the effects of maternal in uteroiAs exposure on weight, total body fat, behavior, and glucose tolerance
at multiple time points into adulthood in both sexes of the F1 and F2
generations. We found arsenic causes physiological metabolic changes in
a sex-, dose-, and generation-specific manner. Further, we found
differentially methylated CpGs (DMCs) and differentially methylated
regions (DMRs) that were unique or persisted between sex, dose, and
generations, identifying DMCs within genes associated T2D or obesity.