Roberta M. Johnke

Radioprotective Effect of American Ginseng on Human Lymphocytes at 90 Minutes Postirradiation: A Study of 40 Cases

BACKGROUND Ionizing radiation (IR) initiates intracellular oxidative stress through enhanced formation of reactive oxygen species (ROS) that attack DNA leading to cell death. Because of the diversity of IR applied in medicine, agriculture, industry, and the growing threats of global terrorism, the acquisition of radioprotectors is an urgent need for the nation. However, the applicability of radioprotectors currently under investigation is limited due to their inherent toxicity. OBJECTIVE This study investigated the effect of a standardized North American ginseng extract (NAGE, total ginsenoside content: 11.7%) on DNA damage in human lymphocytes at 90 minutes postirradiation. DESIGN With the application of NAGE (250-1000 microg mL(-1)) at 90 minutes postirradiation (1 and 2 Gy), DNA damage in lymphocytes obtained from 40 healthy individuals was evaluated by cytokinesis-block micronucleus assay. Similar experiments were also performed in lymphocytes treated with WR-1065 (1 mmol/L or 3 mmol/L). In addition, before and after irradiation, lymphocytes obtained from 10 individuals were measured for their total antioxidant capacity (TAC) and the reactive oxygen species (ROS). RESULTS The significant effect of NAGE against (137)Cs-induced micronuclei (MN) in lymphocytes is concentration dependent. NAGE (750 microg mL(-1)) reduced MN yield by 50.7% after 1 Gy and 35.9% after 2 Gy exposures, respectively; these results were comparable to that of WR-1065. Furthermore, we also found that NAGE reduces MN yield and ROS but increases TAC in lymphocytes. CONCLUSIONS Our results suggest that NAGE is a relatively nontoxic natural compound that holds radioprotective potential in human lymphocytes even when applied at 90 minutes postirradiation. One of the radioprotective mechanisms may be mediated through the scavenging of free radicals and enhancement of the intracellular TAC.

Radioprotective agents for radiation therapy: future trends

Only two radioprotective compounds, amifostine and palifermin, currently have the US FDA approval for use in radiation therapy. However, several agents have been reported that show therapeutic promise. Many of these agents are free radical scavengers/antioxidants. Superoxide dismutase and superoxide dismutase mimetics, nitroxides and dietary antioxidants are all being investigated. Recently, alternative strategies of drug development have been evolving, which focus on targeting the series of cellular insult recognition/repair responses initiated following radiation. These agents, which include cytokines/growth factors, angiotensin-converting enzyme inhibitors and apoptotic modulators, show promise of having significant impact on the mitigation of radiation injury. Herein, we review current literature on the development of radioprotectors with emphasis on compounds with proven or potential usefulness in radiation therapy.

Improved Methods for Extracting RNA from Exfoliated Human Colonocytes in Stool and RT-PCR Analysis

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

A generalized linear-quadratic model incorporating reciprocal time pattern of radiation damage repair

PURPOSE It has been conventionally assumed that the repair rate for sublethal damage (SLD) remains constant during the entire radiation course. However, increasing evidence from animal studies suggest that this may not the case. Rather, it appears that the repair rate for radiation-induced SLD slows down with increasing time. Such a slowdown in repair would suggest that the exponential repair pattern would not necessarily accurately predict repair process. As a result, the purpose of this study was to investigate a new generalized linear-quadratic (LQ) model incorporating a repair pattern with reciprocal time. The new formulas were tested with published experimental data. METHODS The LQ model has been widely used in radiation therapy, and the parameter G in the surviving fraction represents the repair process of sublethal damage with T(r) as the repair half-time. When a reciprocal pattern of repair process was adopted, a closed form of G was derived analytically for arbitrary radiation schemes. The published animal data adopted to test the reciprocal formulas. RESULTS A generalized LQ model to describe the repair process in a reciprocal pattern was obtained. Subsequently, formulas for special cases were derived from this general form. The reciprocal model showed a better fit to the animal data than the exponential model, particularly for the ED50 data (reduced χ(2) (min) of 2.0 vs 4.3, p = 0.11 vs 0.006), with the following gLQ parameters: α/β = 2.6-4.8 Gy, T(r) = 3.2-3.9 h for rat feet skin, and α/β = 0.9 Gy, T(r) = 1.1 h for rat spinal cord. CONCLUSIONS These results of repair process following a reciprocal time suggest that the generalized LQ model incorporating the reciprocal time of sublethal damage repair shows a better fit than the exponential repair model. These formulas can be used to analyze the experimental and clinical data, where a slowing-down repair process appears during the course of radiation therapy.

American Ginseng Modifies 137Cs-Induced DNA Damage and Oxidative Stress in Human Lymphocytes

The multifold bioactive medicinal properties of ginseng have been closely linked to its antioxidative ability, which is related to its ginsenoside content. Since the key mechanism of radiation-induced cell death and tissue damage is the generation of reactive oxygen species (ROS) that attack cellular DNA, this study focuses on the impact of a standardized North American ginseng extract (NAGE) on (137)Cs-induced oxidative stress in human peripheral lymphocytes (PBL) obtained from 10 healthy individuals (6M/4F), 42.7 +/- 4.6 years of age. At two different time points (0 h and 24 h before irradiation), we applied NAGE (250 - 1000 microg ml(-1)) to mononuclear cell cultures for cytokinesis-block micronuclei (MN) assay and determination of the state of oxidative stress in PBL. We found that at both time points, NAGE significantly reduced the MN yields in PBL after irradiation (1 and 2 Gy) in a concentration-dependent manner (P<0.001). Compared with radiation alone, the maximum reduction rate of MN yield were 51.1% and 49.1% after 1 Gy and 2 Gy exposures, respectively. We also found that before irradiation the presence of NAGE in the culture medium resulted in a significant increased intracellular total antioxidant capacity (TAC) in PBL. At both time points, the increment of (137)Cs-induced MN yields in PBL was positively correlated with the increment of intracellular ROS production (R = 0.6 - 0.7, P = 0.002), but negatively correlated with the reduction of TAC levels (R = -0.4 -0.5, P = 0.02 - 0.004). However, the presence of NAGE in the culture medium significantly increased the TAC levels, while concomitantly decreasing both ROS production and MN yields in PBL (P<0.001). Our findings that NAGE is effective in protecting human PBL against radiation-induced oxidative stress should encourage further in vivo study of dietary supplementation with NAGE as an effective natural radiation countermeasure.

Effect of North American Ginseng on 137Cs-induced Micronuclei in Human Lymphocytes : A Comparison with WR-1065

To explore the radioprotective effect of a standardized North American ginseng extract (NAGE) on human peripheral blood lymphocytes (PBL), a micronuclei (MN) assay was conducted in PBL obtained from 12 volunteers. NAGE (50–1000 µg/mL) and WR‐1065 (1 mm and 3 mm) were applied to PBL cultures at 0 h and 90 min post‐irradiation. It was found that (1) the baseline MN yield of PBL ranged from 14.4 ± 1.5 to 15.9 ± 1.5 per 1000 binucleated cells (p > 0.05); after irradiation (1 Gy and 2 Gy), the MN yield increased sharply; (2) MN yields declined with increasing concentrations of NAGE and WR‐1065. Even at 90 min post‐irradiation of 1 Gy, the maximum level of MN reduction rate caused by NAGE and WR‐1065 was 53.8% and 59.2%, respectively; after 2 Gy irradiation, it was 37.3% and 42%, respectively; (3) the MN distribution in PBL followed a non‐Poisson distribution in all cases; and (4) both NAGE and WR‐1065 showed no significant effect on the proliferation index of lymphocytes. The results indicate that NAGE is a relatively non‐toxic natural product, which can be administered as a dietary supplement and has the potential to be a radiation countermeasure. Copyright