Radioprotective Agents To Prevent Cellular Damage Due To Ionizing Radiation
Mar 11, 2022
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Tyler A. Smith1, Daniel R. Kirkpatrick2, Sean Smith2, Trevor K. Smith3, Tate Pearson4, Aparna Kailasam2, Kortney Z. Herrmann4, Johanna Schubertv2 and Devendra K. Agrawal2*
Medical imaging has become a central component of modern medical diagnosis. Over the past 10 years, increased utilization of X-ray examinations and computed tomography (CT) has led to corresponding increases in patient exposure to ionizing radiation raising awareness of the public to its deleterious effects. Despite notable decreases in the radiation dose associated with individual scans, increased utilization of medical imaging is a major contributor to radiation exposure and radiation-associated pathology [1, 2]. Long-term studies of the Second World War atomic bomb survivors in Japan; i.e. those with significant radiation exposure, have been found to have an increased incidence of both leukemia and solid cancers [1]. Based on the linear no-threshold model, imaging-related radiation, while certainly less dramatic than an atomic explosion, may pose significant radiation-related risks. The risks associated with radiation exposure are known to be more pronounced in younger patients. This fact is demonstrated by the increased prevalence of leukemia and solid tumors among the pediatric atomic bomb survivors compared to those who underwent the same radiation exposure at an older age [1]. Gilbert et al. [2] showed a dose-dependent relationship between radiation exposure and leukemia, breast cancer, thyroid cancer, and other solid tumors. Ionizing radiation has immediate, measurable deleterious effects on cells, including increasing reactive oxygen species (ROS), generation of single-stranded DNA breaks (SSBs), and double-stranded DNA breaks (DSBs)
Several authors have proposed using a variety of agents to modulate the cellular damage associated with radiation exposure. It is postulated, for example, that antioxidants or glutathione-elevating compounds may be able to reduce DNA damage, theoretically reducing carcinogenesis post-radiation [4, 5]. Although many studies have demonstrated potential benefits for a variety of agent radioprotective compounds are not routinely administered to patients before or after medical imaging [6]. The aim of this review is to summarize and critically evaluate the recently published findings in the literature that investigated the use of radioprotective agents to avoid radiation-associated cell damage.
Ionizing radiation is widely used in medical diagnostics, cancer-related therapy, and has additional industrial applications [7]. Known hazards associated with human exposure to ionizing radiation include induction of cellular death, genetic mutations, and carcinogenesis [7]. In addition to direct cellular effects, radiation exposure can also damage cells through the generation of reactive oxygen species (i.e. hydrogen peroxide, lipid hydroperoxides, superoxide, hydroxide, hydride, and peroxynitrite). Reactive oxygen species (ROS) are formed when ionizing radiation is absorbed by small molecules, primarily water, surrounding cellular bio-macromolecules. These ROS react with cellular contents, including DNA and proteins [7].
The cell responds to increased concentrations of free radicals by generating natural antioxidants (including superoxide dismutase, glutathione, catalase) which can minimize or eliminate free-radical induced damage to cellular structures. Glutathione peroxidase primarily catalyzes the conversion of hydroxide ions to water. Superoxide dismutase converts superoxides to hydrogen peroxide, which is then converted to oxygen and water by catalase. Superoxide dismutase exists in several different isoforms, each of which is specialized to specific areas of the cell [8]. When exposed to increasing levels of ionizing radiation, the cell increases the expression of antioxidant enzymes [8]. When, however, the level of ROS overwhelms these cellular defenses, the cell will sustain damage (in a dose-dependent manner) that can lead to carcinogenesis, teratogenesis, necrosis, or apoptosis.
Administering radioprotective agents has been proposed as one way to decrease radiation-related deleterious effects on cells. Antioxidants have the potential to act as free radical scavengers, and thus reduce some DNA damage caused by ionizing radiation [4, 7, 9, 10]. Theoretically, this intervention would allow cellular defenses to keep pace with the free radicals generated by radiation exposure (assuming the intracellular level of anti-oxidants is sufficient at the time of radiation exposure). Radioprotective compounds may suppress free radical formation, remove free radicals, induce natural radioprotector production (such as superoxide dismutase, glutathione peroxidase, and catalase), enhance DNA repair, reduce the post-radiation inflammatory response, or even delay cellular division allowing more time for cells to repair or undergo apoptosis [10] (Table 1). Although radioprotective substances have been shown to be effective at decreasing the side effects of radiation therapy, there are currently no radioprotectants used in diagnostic radiology.
To summarize existing candidates for clinical radioprotectors, we conducted a literature review using a Pubmed/MEDLINE search with key phrases including: “antioxidants in medical imaging”, “antioxidants in radiotherapy”, “antioxidant radiation”, “radioprotective agents”, “radioprotective radiotherapy”, “radioprotective medical imaging”, and “radioprotection.” To be included, the articles were required to be peer-reviewed primary research articles published in the past 10 years that investigated one or more substances as potential radioprotective agents. This article represents a summary and critical analysis of the selected articles investigating radioprotection. Moreover, this article summarizes key findings relevant to the following clinical question: can radioprotectants be used in diagnostic imaging to reduce DNA damage?
Findings from in vitro studies- In vitro: human lymphocytes
The preponderance of the literature on radioprotective agents comes from studying human lymphocytes in vitro before and after exposure to radiation. Occasionally, this includes taking blood samples from patients after they undergo clinical imaging. Usually, these studies quantify radiation-induced double-stranded DNA breaks (DSBs) via γ-H2AX foci (γH2AX). For example, Brand et al. [9] showed that several antioxidants, if administered before exposing human blood to radiation, could lower the incidence of DSBs in lymphocytes. Importantly, N-acetyl cysteine (NAC) and vitamin C lowered DSBs by 43 and 25% respectively, which was significantly more than any of the other agents studied [9]. Interestingly, despite individual agents showing promise as radioprotectants, none of the combinations tested by the authors showed an additive effect when multiple agents were used together [9]. This study supports using antioxidants, particularly NAC and vitamin C analogs, to prevent radiation-associated DNA damage. Like Brand et al. [9], Kuefner et al. [11] conducted a study investigating the effects of a mix of antioxidants (calcium ascorbate, d-alpha-tocopheryl succinate, carotenoids, NAC, R-α-lipoic acid, l-selenomethionine) on in vitro human lymphocytes. Kuefner et al. [11] did this in two ways: first, in vitro lymphocytes were treated with antioxidants, then irradiated. Second, blood samples were obtained 15, 30, 60 min, 2, 3, 5 h after ingestion of a pill contain- ing the study antioxidants, then the lymphocytes were irradiated. While administering antioxidants after irradiation did not lead to a reduction in DSBs, pretreatment with antioxidants caused significant reductions in DSBs, with a 23% reduction after 15 min and a 58% reduction if administered 60 min before irradiation [11]. This study had clinical value because the experimental radiation exposure was comparable to that received during a CT scan. In another study, NAC and vitamin C were both administered immediately before and after patients were exposed to X-rays. The patient's blood was then drawn and DSBs were measured in lymphocytes. Both vitamin C and NAC were found to decrease DSBs, as measured by γH2AX, compared to controls [12]. In each of these studies, NAC significantly decreased radiation-related DNA damage in human lymphocytes.
An important study by Reliene et al. [13] looked at the effects of NAC in human, murine, and yeast models. While this study did find that NAC reduced γH2AX foci (a surrogate for DSBs), it also noted that cell colony survival was unchanged in yeast and human lymphocytes In other words, while NAC decreases DNA damage, it does not necessarily prevent apoptosis or necrosis. This finding may have important potential implications: NAC may decrease the incidence of DNA damage without interfering with the purposeful death of cancerous or pre-cancerous cells [13]. NACs' ability to decrease or avoid DNA damage without protecting cells from apoptosis may increase its clinical value (relative to other antioxidants).
Other studies have focused specifically on vitamin C and its derivatives. In a 2014 study, Xiao et al. [3] exposed human lymphocytes to radiation after being plated for 3 h with differing concentrations of one of two vitamin C derivatives: 6-O-palmitoyl ascorbate (PlmtVC) or l-ascorbic acid (l-AA). As a radioprotective agent, PlmtVC outperformed l-AA, showing that not all vitamin C derivatives are equally efficacious as antioxidants [3]. PlmtVC significantly decreased lipid peroxidation and protein carbonylation compared to controls while also elevating endogenous glutathione [3]. PlmtVC also significantly reduced the total number of DSBs compared with either controls or l-AA [3]. While some studies have shown vitamin C to have radio-protective activity, other studies have shown vitamin C to potentiate radiation-induced damage [14–16]. This dichotomy makes vitamin C a controversial agent for clinical use as a radioprotectant. Although vitamin C and NAC have shown promising results, a multitude of other agents has been studied using human lymphocytes in vitro. Alcaraz et al. [17] conducted a study to assess 10 different antioxidant compounds (carnosic acid, green tea extract, apigenin, diosmin, rosmarinic acid, l-ascorbic acid, δ-tocopherol, rutin, amifostine, dimethylsulphoxide) as candidates for radioprotectants against chromosomal damage caused by ionizing radiation (with DSMO as the control and vehicle). When compared to irradiated controls, all compounds showed a decrease in DNA damage, with the greatest effects seen in rosmarinic acid, carnosic acid, δ-tocopherol (vitamin E), and apigenin [17]. Less effective agents included l-ascorbic acid, amifostine, green tea extract, rutin, and diosmin [17]. This same pattern was also seen in terms of the magnitude of radioprotection provided by these agents [17].
Arivalagan et al. [18] investigated carvacrol (CVC) as a potential radioprotective agent due to its safety for consumption (it is a common food additive), anti-inflammatory, and antioxidant properties. In this study, lymphocytes were collected from healthy individuals and then treated with DMSO or CVC prior to radiation. Not surprisingly, as radiation dose increased cell survival decreased and DNA damage increased in the control groups [18]. Lymphocytes pretreated with CVC experienced a statistically significant rise in the lethal dose of radiation they could tolerate compared to controls. CVC- treated lymphocytes also showed a significant decrease in DNA damage as well as decreased lipid peroxidation and apoptosis [18]. CVC appears to decrease free radical damage in two ways: as an antioxidant and as a free radical scavenger [18]. CVC holds promise as a radioprotective agent with few side effects or toxicity.
Phenolic glycosides, which occur naturally in plants, have also been shown to have antioxidant properties [19]. Materska et al. [19] investigated sev- eral phenolic glycosides: sinapoyl-E-glucoside (sEg), quercetin-3-O-rhamnoside-7-O-glucoside (q3Or7Og), quercetin-3-O-rhamnoside (q3Or) and luteolin-7-O-(2- apiosyl)-glucoside (l7O2ag). The authors used human lymphocytes obtained from healthy human donors, and then exposed them to one of the phenolic glycosides before irradiation with X-rays. Researchers found that q3Or showed the highest radioprotective effect, with a 50% reduction in DNA damage compared to controls. Importantly, in this study, these substances did not show any toxic effects against human lymphocytes [19]. The phenolic glycosides were also noted to have excellent antiradical activities [19]. In this study, compounds with greater superoxide radical scavenging capabilities also demonstrated better radioprotective effects [19]. The radioprotective effects of other phenolic glycosides including quinic and chlorogenic acid have also been studied on human lymphocytes in vitro. In one study, lymphocytes were exposed to different doses of X-ray radiation and treated with different concentrations of either quinic acid, chlorogenic acid, or sham control. This study found that lymphocytes pretreated with both quinic acid and chlorogenic acid prior to irradiation had significant decreases in DNA damage as measured by the genetic damage index [20]. In the case of chloro- genic acid, however, there were no significant changes in the genetic damage index in the lower X-ray radiation dose range [20]. Quinic acid also decreased the percentage of cells damaged by radiation [20]. Quantitatively, the magnitude of protection (based on the genetic damage index) was calculated to be 5.99–53.57% for quinic acid and 4.49–48.15% for chlorogenic acid [20]. The radioprotective efficacy of quinic acid and chlorogenic acid seems to be comparable to other phenolic phytochemicals like curcumin, caffeic acid, hesperidin, vanilla, and resveratrol [20]. The observed effects of both quinic acid and chlorogenic acid may be related to vicinal hydroxyl groups on an aromatic residue which may possess anti-mutagenic, anti-carcinogenic, and antioxidant effects in vitro [20].
Cinnamic acid is a phenolic substance obtained from cinnamon oil and has been shown to have antioxidant properties. Cinkilic et al. [21] investigated the radioprotective effects of cinnamic acid against X-ray-induced genomic instability in human lymphocytes. They found that cinnamic acid-treated lymphocytes had a significant decrease in DNA DSBs (range from 16 to 55% reduction) compared to controls [21]. Pretreatment with cinnamic acid also reduced total genetic damage [21]. Cinnamic acid alone did not increase DSBs or other DNA damage, suggesting it is not genotoxic [21]. The authors found that cinnamic acid decreased DNA damage induced by irradiation with X-rays by reducing the intracellular ROS level through its free-radical scavenging properties [21]. As a group, phenolic glycosides include many agents that show potential for decreasing radiation-associated DNA damage.
