In this ex vivo study of PBL obtained from 40 healthy human subjects, we have demonstrated the potential of a standardized NAGE and WR-1065 to modulate 137Cs-induced oxidative stress in PBL at 90 minutes after exposure, thereby indicating their postexposure radioprotective effect. The cell membrane of PBL has very high phospholipid content, rendering PBL vulnerable to oxidative damage.7 MN formation in PBL is a well-established biomarker for radiation-induced damage and free-radical impacts.22 We irradiated PBL with 1–2 Gy, because this dose range induces significant DNA damage, without the possibility of instant killing or causing selective interphase cell death.
The radioprotective effect and the antioxidative potentials of ginseng are no longer novel findings;15,18,23,24 nevertheless, to the best of our knowledge, no published data have ever been used to analyze the effect of NAGE on intracellular oxidant–antioxidant homeostasis in human PBL after irradiation. We discovered three interesting findings in this study. First, at 90 minutes postirradiation, both NAGE and WR-1065 significantly reduced the 137Cs-induced MN yields in a concentration-dependent manner (Tables 1 and and2).2). This relatively long postirradiation protective window of the two agents could be of potential clinical interest if it is upheld in in vivo studies.
Second, we also found that at 90 minutes postirradiation, the maximum reduction rates by NAGE (750μg mL−1) and WR (3mmol/L) of MN yields in PBL were 50.7% and 52% after 1 Gy and 35.9% and 33.4% after 2 Gy irradiation, respectively (Table 1). These results suggest that the potential of NAGE against radiation-induced MN production in PBL is comparable to that of WR-1065. In addition, at concentrations up to 1000μg mL−1 (NAGE) or 3mmol/L (WR-1065), respectively, no modulation of MN yield was found in PBL before irradiation (Table 1), indicating that this protection is accomplished without apparent genomic toxicity in PBL.
Third, we further observed in PBL obtained from 10 human subjects that the rise in 137Cs-induced MN yields in PBL is paralleled by the ROS level at the time of irradiation in a radiation dose-dependent manner (p=0.03–0.005), but is inversely correlated with the reduction of TAC (p=0.005–0.001) (Fig. 1A–C). This finding suggests that the existing endogenous antioxidants in PBL were not able to counteract the 137Cs-induced oxidative stress, and thus the increased MN production was a function of ROS accumulation and DNA damage. However, with the application of NAGE to the culture medium (250–1000μg mL−1) at 90 minutes postirradiation, the observed significant increase in intracellular TAC level and concomitant decrease of both MN yield and ROS level strongly indicate a restoration of antioxidant capacity in PBL by NAGE treatment.
Both ginseng and WR-1065 confer radioprotection by scavenging IR-induced ROS. However, the exact mechanism underlying the 90 minutes postexposure radioprotective effect of NAGE is unclear. It may be related to the upregulation of antioxidant enzymes induced by NAGE at the level of gene expression 90 minutes postexposure.8 As demonstrated in vitro,19,25,26 the delayed radioprotection of WR-1065 is associated with the effect of manganese superoxide dismutase (SOD2). Since the molecular components of ginseng responsible for this scavenging action are ginsenosides,7,8 the radioprotective potential of ginseng is likely directly related to its ginsenoside content, which is quite high in our NAGE formulation (11.7%). Ginsenosides in NAGE are capable of intercalating in the plasma membrane, leading to changes in membrane fluidity and eliciting a cellular response to IR-induced cytotoxic stress.27In this study, we found that the application of NAGE to the culture medium at 90 minutes post 137Cs exposure significantly increased intracellular TAC levels and was accompanied by a significant decrease in both ROS and MN yields in PBL (Fig. 1). Under our experimental design, the intracellular TAC in PBL represents the cumulative antioxidant capacity, including both NAGE-derived antioxidants and those of endogenous origin. Our findings suggest that (1) the lipid-soluble and water-soluble antioxidant of NAGE permeates into PBL and suppresses 137Cs-induced MN and ROS via OH radical scavenging; and (2) this action could be occurring directly through free-radical scavenging or indirectly through upregulation of antioxidant enzymes.10,28 Based on our findings concerning antioxidant activity at the cellular level, it appears that the postexposure protection of NAGE may be due either to its potential for modulating the redox homeostasis or for boosting the intracellular antioxidant defense system in human PBL.
Our findings are in agreement with the belief that supplementation of antioxidants could inhibit the ROS-induced DNA damage in human PBL.13 However, our results were generated from ex vivo experiments after up to 2 Gy irradiation of PBL. Also, the antioxidant capacities of NAGE measured ex vivo may not be consistent with their effects in vivo. For instance, after oral ingestion of ginseng, both gastric digestion and hydrolysis by intestinal microflora lead to the biotransformation of ginsenosides.29 Subsequently, ginsenoside metabolites can be absorbed into the blood, whence they can exert their active pharmacological effects. Since intestinal bacteria are sensitive to host conditions, the individual physiologic variations in bacteria-hydrolyzing potentials may affect the radioprotective efficiency of NAGE. Thus, the clinical relevance of our findings that the concentration of NAGE at 750μg mL−1 reduced both MN yields and ROS levels in human PBL ex vivo (Table 1, Fig. 1) would be hard to predict. We are planning to answer these questions in a future research project.
Currently, amifostine (WR-2721) is the only radioprotective agent approved by the U.S. Food and Drug Administration for cancer patients undergoing radiotherapy. However, the limitations associated with amifostine include its inherent toxicity, high cost, intravenous administration route to be applied 15 minutes before radiotherapy, and possible tumor protection.1,2,30 In contrast, NAGE is a relatively nontoxic, inexpensive natural product with broad medicinal and pharmacological activities, including antitumor activity that can be orally administered under emergency conditions or as a dietary supplement. We believe, therefore, that NAGE is a candidate eminently suited for addition to the list of potential radioprotectors.