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.