Changes in one-carbon metabolism and DNA methylation in the hearts of mice exposed to space environment-relevant doses of oxygen ions (¹⁶O)

Highlights

• Space-relevant doses of radiation lead to long-lasting changes in DNA methylation.

• Major satellites are particularly affected, at the methylation and expression levels.

• Transsulfuration of methionine is impacted at low dose.

Abstract

Cardiovascular disease constitutes an important threat to humans after space missions beyond the Earth's magnetosphere. Epigenetic alterations have an important role in the etiology and pathogenesis of cardiovascular disease. Previous research in animal models has shown that protons and ⁵⁶Fe ions cause long-term changes in DNA methylation and expression of repetitive elements in the heart. However, astronauts will be exposed to a variety of ions, including the smaller fragmented products of heavy ions after they interact with the walls of the space craft. Here, we investigated the effects of ¹⁶O on the cardiac methylome and one-carbon metabolism in male C57BL/6 J mice. Left ventricles were examined 14 and 90 days after exposure to space-relevant doses of 0.1, 0.25, or 1 Gy of ¹⁶O (600 MeV/n). At 14 days, the two higher radiation doses elicited global DNA hypomethylation in the 5′-UTR of Long Interspersed Nuclear Elements 1 (LINE-1) compared to unirradiated, sham-treated mice, whereas specific LINE-1 elements exhibited hypermethylation at day 90. The pericentromeric major satellites were affected both at the DNA methylation and expression levels at the lowest radiation dose. DNA methylation was elevated, particularly after 90 days, while expression showed first a decrease followed by an increase in transcript abundance. Metabolomics analysis revealed that metabolites involved in homocysteine remethylation, central to DNA methylation, were unaffected by radiation, but the transsulfuration pathway was impacted after 90 days, with a large increase in cystathione levels at the lowest dose. In summary, we observed dynamic changes in the cardiac epigenome and metabolome three months after exposure to a single low dose of oxygen ions.

Introduction

The potential health hazards associated with space radiation are a major obstacle preventing deep space exploration (Chancellor et al., 2014). Beyond the protective magnetosphere of the Earth, astronauts are exposed to radiation from galactic cosmic rays and solar flares. For even the shortest round-trip to Mars, exposure for the crew is estimated to reach 0.66 Sv (or Gy) (Zeitlin et al., 2013). Furthermore, contrary to terrestrial radiation, current technology does not provide effective shielding against the type of high energy particles encountered in space. It is therefore crucial to learn more about the types of long-term biological changes associated with exploration beyond the orbit of the Earth.

Experimental evidence suggests cardiac remodeling, as well as apoptotic and inflammatory responses in the mouse heart as a result of exposure to high-LET radiation (Boerma et al., 2016, Ramadan et al., 2016, Tungjai et al., 2013, Boerma et al., 2015, Hughson et al., 2018). Studies of cancer survivors indicate that cardiac injury due to exposure to ionizing radiation increases linearly with mean dose of radiation to the heart, and progresses very slowly, with onset observed decades after exposure (Darby et al., 2013). Recent studies indicate that epigenetic and metabolic alterations may substantially contribute to pathogenesis of cardiovascular disease (Stenvinkel et al., 2007, Sharma et al., 2008). DNA methylation is one of the key epigenetic mechanisms for the regulation of genetic information. It is responsive to environmental exposures, providing a possible mechanism linking radiation exposure and late-onset injury to the heart.

Methyl groups for DNA methylation are supplied through the methyl donor methionine, with homocysteine as a byproduct. The methionine cycle is critical for normal heart development, and folic acid supplementation during pregnancy is associated with a decreased incidence of congenital heart defects (Feng et al., 2015, Liu et al., 2016). Beyond the development period, an elevated level of homocysteine is an independent risk factor for cardiovascular disease (Perry et al., 1995, Wald et al., 2011), highlighting the importance of the methionine cycle in cardiac health throughout life.

Studies report that radiation-induced changes in DNA methylation stem primarily from the repetitive elements rather than individual genes (Nzabarushimana et al., 2014, Miousse et al., 2017b, Koturbash et al., 2016). Of particular interest are two subtypes of repetitive elements; LINE-1 retrotransposons and satellite DNA. LINE-1 repeats cover about 20% of the murine genome and are heavily methylated within their 5′-untranslated regions (UTRs) (Miousse and Koturbash, 2015). Satellite DNA repeats are characteristic of the pericentromeric regions. It is a less abundant, but still heavily methylated type of repetitive element. DNA methylation is a critical regulator of aberrant transcription for both elements, and DNA hypomethylation is associated with their reactivation and insertional mutagenesis (for LINE-1 elements) and chromosomal aberrations (for satellite DNA) (Miousse and Koturbash, 2015, Miousse et al., 2015b, Koturbash et al., 2007, Miousse et al., 2015a, Qu et al., 1999, Ji et al., 1997). Recent studies demonstrated alterations in DNA methylation during the pathogenesis of a number of heart diseases (Baccarelli et al., 2010, Kim et al., 2010, Gilsbach et al., 2014, Fiorito et al., 2014). DNA hypomethylation and accumulation of satellite DNA transcripts has also been observed in humans diagnosed with heart failure (Haider et al., 2012). Effects of high-LET irradiation on DNA methylation of those two types of repetitive elements in the mouse heart, as well as alterations to one-carbon metabolism, were reported (Koturbash et al., 2016).

In this study, we investigate the short- and long-term (14 and 90 days post exposure, respectively) effects of exposure to one of the under-investigated heavy ions yet relevant for the radiation environment inside a space craft, oxygen (¹⁶O), on the mouse cardiac methionine cycle using epigenetic and metabolomic approaches. The most biologically relevant radiation source in free space is ⁵⁶Fe (Cucinotta et al., 2003), and we previously reported epigenetic changes in the heart associated with this type of radiation (Koturbash et al., 2016). However, interaction with the hull of the spacecraft and space suit materials also creates ions of smaller masses, such as ¹⁶O (Walker et al., 2013). Previous results from our group and others indicate a distinctly large cardiovascular toxicity associated with exposure to ¹⁶O, which prompted us to take a closer look at the epigenetic signature of ¹⁶O in the heart (Ramadan et al., 2016, Yan et al., 2014, Seawright et al., 2019). Levels of radiation exposure have been estimated for different types of charged particles in outer space and on the surface of Mars (Nelson, 2016, Cucinotta et al., 2003, Zeitlin et al., 2013). Several Russian, European and Canadian space agencies have proposed 1 Sv (or 1 Gy) as the astronaut career exposure limit, a dose only slightly above the high estimates for the cumulative exposure associated with a round trip mission to Mars. Based on these estimates, we analyzed doses of 0.1, 0.25, and 1 Gy of ¹⁶O radiation (600 MeV/n) in an effort to replicate the most current estimation of exposures associated with a round trip to Mars, but with all of the dose coming from a single particle species.