The impact of ionizing radiation on placental trophoblasts

Abstract

Introduction

Exposure to low-dose radiation is widespread and attributable to natural sources. However, occupational, medical, accidental, and terrorist-related exposures remain a significant threat. Information on radiation injury to the feto-placental unit is scant and largely observational. We hypothesized that radiation causes trophoblast injury, and alters the expression of injury-related transcripts in vitro or in vivo, thus affecting fetal growth.

Methods

Primary human trophoblasts (PHTs), BeWo or NCCIT cells were irradiated in vitro, and cell number and viability were determined. Pregnant C57Bl/6HNsd mice were externally irradiated on E13.5, and placentas examined on E17.5. RNA expression was analyzed using microarrays and RT-qPCR. The experiments were repeated in the presence of the gramicidin S (GS)-derived nitroxide JP4-039, used to mitigate radiation-induced cell injury.

Results

We found that survival of in vitro–irradiated PHT cell was better than that of irradiated BeWo trophoblast cell line or the radiosensitive NCCIT mixed germ cell tumor line. Radiation altered the expression of several trophoblast genes, with a most dramatic effect on CDKN1A (p21, CIP1). Mice exposed to radiation at E13.5 exhibited a 25% reduction in mean weight by E17.5, and a 9% reduction in placental weight, which was associated with relatively small changes in placental gene expression. JP4-039 had a minimal effect on feto-placental growth or on gene expression in irradiated PHT cells or mouse placenta.

Discussion and conclusion

While radiation affects placental trophoblasts, the established placenta is fairly resistant to radiation, and changes in this tissue may not fully account for fetal growth restriction induced by ionizing radiation.

Introduction

Exposure to ionizing radiation remains a reality in today's world. Worldwide, the average annual exposure to natural radiation is about 2.4 milli Sievert (mSv) [1]. Occupational exposures are most relevant to people working with nuclear fuel and medical devices, in defense-related functions, and in occupations associated with enhanced exposure to natural sources of radiation. For example, aircrew members are exposed to 5–8 mSv per hour while flying [1]. Medical sources of radiation include diagnostic procedures that expose individuals to low doses (commonly 0.1–10 mSv) and therapeutic exposures, typically 20–60 Gray (Gy), to a targeted tissue [1]. Accidental exposures in nuclear fuel processing plants typically expose workers to 1–20 Gy [1]. These risks may be greatly amplified if “dirty bombs” are deployed by terrorists against civilians [2].

Research into diagnosis, treatment, and prevention of radiation injury in pregnancy is limited by appropriate ethical concerns and by the scarcity of information on mechanisms underlying the effect of ionizing radiation on the developing feto-placental unit. Anecdotal reports or observational studies have generated some information pertaining to gestational age and radiation dose. During the pre-implantation period, as little as 0.3 Gy is lethal to the mouse embryo [3]. In the post-implantation period, the main risks from radiation include embryonic death, congenital anomalies, growth restriction, and neurologic maldevelopment [4]. Exposing mice at E14 to 0.3–1.5 Gy of whole body irradiation caused decreased neonatal body length and body weight [5]. Minimal effects on litter size or fetal growth were observed when mice at E7-16 were exposed to low dose radiation, 10–13 mSv per day over 10 days [6]. In humans, data from children exposed to in utero radiation after catastrophic events in Hiroshima, Nagasaki, and Chernobyl revealed lower height and weight in adolescence [4], [7].

Ionizing radiation damages tissues through diverse mechanisms [8]. A major consequence of radiation is direct and indirect DNA damage. Direct effects include the transfer of kinetic energy from radioactive particles to the DNA backbone, which breaks phosphodiester bonds. Indirect effects include the generation of reactive oxygen species, which cause DNA double-strand breaks and cell-cycle arrest. Other types of injury include p53-dependent and -independent apoptosis [9], mitochondrial damage, loss of regenerative capacity, and premature senescence [8]. NFκB mediates several radiation-stimulated signal transduction pathways, which may explain the degree of radiation-sensitivity of differing cells types [10]. These pathways implicate CDKN1A (also known as p21, CIP1), epidermal growth factor receptors, and the apoptosis-related proteins BAX and BCL2 in radiation injury [11]. Whereas radiation-induced pathways have been interrogated in non-placental cell types, there are no studies of radiation injury to cultured primary human trophoblast (PHT) cells; there has been a single study that included the choriocarcinoma line JEG3 and showed no effect on gene expression of gap junction protein alpha 1 [12].

Methods to scavenge reactive oxygen species have been proposed to mitigate radiation damage. This effect has been attributed, at least in part, to the action of manganese superoxide dismutase (MnSOD, [13]). The nitroxides, which have superoxide dismutase–mimetic activity and inhibit lipid peroxidation [14], constitute one such class of radioprotectors. JP4-039 is a nitroxide linked to a short alkene isostere analog of hemigramicidin S, which allows concentration at the mitochondrial membrane, the site of radiation-induced lipid peroxidation [15]. It has been shown to protect against radiation damage in vivo [16], [17]. In this study, we tested the hypothesis that ionizing radiation causes injury to PHT cells in vitro and to the mouse placenta in vivo. We also assessed whether the nitroxide JP4-039 mitigates that damage.