Pentagalloyl Glucose-Targeted Inhibition of P-Glycoprotein and Re-Sensitization of Multidrug-Resistant Leukemic Cells (K562/ADR) to Doxorubicin: In Silico and Functional Studies.

Combining phytochemicals with chemotherapeutic drugs has demonstrated the potential to surmount drug resistance. In this paper, we explore the efficacy of pentagalloyl glucose (PGG) in modulating P-gp and reversing multidrug resistance (MDR) in drug-resistant leukemic cells (K562/ADR). The cytotoxicity of PGG was evaluated using a CCK-8 assay, and cell apoptosis was assessed using flow cytometry. Western blotting was used to analyze protein expression levels. P-glycoprotein (P-gp) activity was evaluated by monitoring the kinetics of P-gp-mediated efflux of pirarubicin (THP). Finally, molecular docking, molecular dynamics simulation, and molecular mechanics with generalized Born and surface area solvation (MM-GBSA) calculation were conducted to investigate drug-protein interactions. We found that PGG selectively induced cytotoxicity in K562/ADR cells and enhanced sensitivity to doxorubicin (DOX), indicating its potential as a reversal agent. PGG reduced the expression of P-gp and its gene transcript levels. Additionally, PGG inhibited P-gp-mediated efflux and increased intracellular drug accumulation in drug-resistant cells. Molecular dynamics simulations and MM-GBSA calculation provided insights into the binding affinity of PGG to P-gp, suggesting that PGG binds tightly to both the substrate and the ATP binding sites of P-gp. These findings support the potential of PGG to target P-gp, reverse drug resistance, and enhance the efficacy of anticancer therapies.

Modulation of p-glycoprotein-mediated efflux pirarubicin in living multidrug-resistant K562/Dox cell lines by 4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid via impairment of the cellular energetic state.

The objective was to investigate the effect of 4-hydroxybenzoic acid (4-HBA) and 4-hydroxy-3-methoxybenzoic acid (Vanillic acid, VA) on p-glycoprotein (P-gp) activity in multidrug-resistant K562/Dox cancer cells. The cytotoxic and co-treatment with pirarubicin (Pira) were analyzed using a resazurin assay. A noninvasive functional spectrofluorometric technique was used to determine the kinetics of Pira uptake in living multidrug-resistant K562/Dox cancer cells. The three biological endpoints for determination of cellular energetic state included the activity of mitochondria, mitochondrial membrane potential (ΔΨm), and ATP levels. The results revealed that 4-HBA (10 mM) and VA (5 and 10 mM) statistically decreased cell viability in K562 and multidrug-resistant K562/Dox cancer cells. In ways consistent with that result, 4-HBA and VA (0.01, 0.1, 1, and 10 mM) could statistically decrease the IC50 of Pira in K562 and multidrug-resistant K562/Dox cancer cells at 48 and 72 h. The overall intracellular Pira concentration increased in 4-HBA- and VA-treated multidrug-resistant K562/Dox cancer cells when compared to control. The ratio of ka i/ka 0 in 4-HBA- and VA-treated multidrug-resistant K562/Dox cancer cells was significantly decreased when 4-HBA and VA concentration increased. The activity of mitochondria, ΔΨm, and ATP levels significantly reduced in multidrug-resistant K562/Dox cancer cells incubated with 0.01, 0.1, 1, and 10 mM 4-HBA and VA at all harvest time points. In conclusion, 4-HBA and VA were able to bring about cell death in multidrug-resistant K562/Dox cancer cell at high concentrations. The 4-HBA and VA could modify P-gp function via an impaired cellular energetic state, resulting in increased in intracellular drug concentration in multidrug-resistant K562/Dox cancer cells.

Effect of pre-low-dose irradiation on anticancer activities of gallic acid in leukemic K562 and K562/Dox cells: cell viability and cellular energetic state studies.

The aim of this study was to determine the effects of pre-low-dose irradiation followed by gallic acid (GA) on cell viability and cellular energetic state of leukemic K562 and K562/Dox cells. The cells were irradiated with 0.02, 0.05, and 0.1 Gy of X-rays. For determining cell viability, pre-low-dose irradiation was followed by 10 or 100 µM GA at 24 h post-irradiation, and the cell viability was then determined at 48 h post-irradiation. For cellular energetic state, pre-low-dose irradiation was followed by 10 or 100 µM GA at 1.5 h post-irradiation and the mitochondrial activity, mitochondrial membrane potential (ΔΨm), and ATP level were determined at 3 h post-irradiation. The % cell viability was significantly decreased in both cells that were irradiated with X-rays followed by treatment with 10 or 100 µM GA at 24 h post-irradiation, when compared with control group. However, this did not happen when compared with GA alone without any pre-low-dose irradiation. The mitochondrial activity had significantly decreased in 10 µM GA-treated K562 cells and the mitochondrial activity, ΔΨm, and ATP levels had significantly decreased in 10 µM GA-treated K562/Dox cells after irradiation to X-rays when compared with GA alone group. In addition, the ΔΨm and ATP levels was significantly decreased in only 100 µM GA-treated K562/Dox cells, but was not decreased in 100 µM GA-treated K562 cells after exposure to X-rays. These findings suggest that pre-low-dose irradiation followed by GA could not kill K562 and K562/Dox cells, but could improve cellular energetic damage of GA effects possibly through mitochondrial impairment.

Advanced Molecular Imaging (MRI/MRS/1H NMR) for Metabolic Information in Young Adults with Health Risk Obesity.

BACKGROUND: Obesity or being overweight is a medical condition of abnormal body fat accumulation which is associated with a higher risk of developing metabolic syndrome. The distinct body fat depots on specific parts of the anatomy have unique metabolic properties and different types of regional excessive fat distribution can be a disease hazard. The aim of this study was to identify the metabolome and molecular imaging phenotypes among a young adult population. METHODS: The amount and distribution of fat and lipid metabolites profile in the abdomen, liver, and calf muscles of 46 normal weight, 17 overweight, and 13 obese participants were acquired using MRI and MR spectroscopy (MRS), respectively. The serum metabolic profile was obtained using proton NMR spectroscopy. NMR spectra were integrated into seven integration regions, which reflect relative metabolites. RESULTS: A significant metabolic disorder symptom appeared in the overweight and obese group, and increased lipid deposition occurred in the abdomen, hepatocytes, and muscles that were statistically significant. Overall, the visceral fat depots had a marked influence on dyslipidemia biomarkers, blood triglyceride (r = 0.592, p < 0.001), and high-density lipoprotein cholesterol (r = -0.484, p < 0.001). Intrahepatocellular lipid was associated with diabetes predictors for hemoglobin (HbA1c%; r = 0.379, p < 0.001) and for fasting blood sugar (r = 0.333, p < 0.05). The lipid signals in serum triglyceride and glucose signals gave similar correspondence to biochemical lipid profiles. CONCLUSIONS: This study proves the association between alteration in metabolome in young adults, which is the key population for early prevention of obesity and metabolic syndrome. This study suggests that dyslipidemia prevalence is influenced mainly by the visceral fat depot, and liver fat depot is a key determinant for glucose metabolism and hyperglycemia. Moreover, noninvasive advanced molecular imaging completely elucidated the impact of fat distribution on the anthropometric and laboratory parameters, especially indices of the metabolic syndrome biomarkers in young adults.

Gallic acid enhances pirarubicin­induced anticancer in living K562 and K562/Dox leukemia cancer cells through cellular energetic state impairment and P­glycoprotein inhibition.

Leukemia is a common malignancy affecting humans worldwide. Pirarubicin (Pira) is one of the anticancer agents used for the treatment of leukemia. Although Pira is effective, drug resistance may develop in cancer cells exposed to this drug, whereas the combination of natural products with Pira may help to overcome this problem. The aim of the present study was to focus on the effect of gallic acid (GA) on the anticancer activity of Pira in K562 leukemia cells and K562/doxorubicin (Dox)­resistant leukemia cells in order to investigate the possible underlying mechanisms. The cell viability, mitochondrial activity, mitochondrial membrane potential (ΔΨm) and ATP levels were assessed in living K562 and K562/Dox cancer cells following treatment with GA/Pira combination, GA alone or Pira alone. P­glycoprotein­mediated efflux of Pira was determined in GA­treated K562/Dox cancer cells. The results demonstrated that GA/Pira combination decreased cell viability, mitochondrial activity, ΔΨm and ATP levels in K562 and K562/Dox cancer cells in a GA concentration­dependent manner compared with non­treated or Pira­treated cells. GA inhibited P­glycoprotein­mediated efflux of Pira in GA­treated K562/Dox cancer cells. Therefore, GA enhanced the anticancer effect of Pira on K562 and K562/Dox cancer cells through cellular energy status impairment, and was able to reverse drug resistance in living K562/Dox cancer cells by inhibiting the function of P­glycoprotein.

Identification of Metabolic Phenotypes in Young Adults with Obesity by 1H NMR Metabolomics of Blood Serum.

(1) Since the obesity prevalence rate has been consistently increasing, it is necessary to find an effective way to prevent and treat it. Although progress is being made to reduce obesity in the young adult population, a better understanding of obesity-related metabolomics and related biochemical mechanisms is urgently needed for developing appropriate screening strategies. Therefore, the aim of this study is to identify the serum metabolic profile associated with young adult obesity and its metabolic phenotypes. (2) Methods: The serum metabolic profile of 30 obese and 30 normal-weight young adults was obtained using proton nuclear magnetic resonance spectroscopy (1H NMR). 1H NMR spectra were integrated into 24 integration regions, which reflect relative metabolites, and were used as statistical variables. (3) Results: The obese group showed increased levels of lipids, glucose, glutamate, N-acetyl glycoprotein, alanine, lactate, 3 hydroxybutyrate and branch chain amino acid (BCAA), and decreased levels of choline as compared with the normal-weight group. Non-hyperlipidemia obese adults showed lower levels of lipids and lactate, glutamate, acetoacetate, N-acetyl glycoprotein, isoleucine, and higher levels of choline and glutamine, as compared with hyperlipidemic obese adults. (4) Conclusions: This study reveals valuable findings in the field of metabolomics and young adult obesity. We propose several serum biomarkers that distinguish between normal weight and obese adults, i.e., glutamine (higher in the normal group, p < 0.05), and lactate, BCAAs, acetoacetate and 3-hydroxybutyrate (higher in the obese group, p < 0.05). In addition, visceral fat and serum TG, glutamate, acetoacetate, N-acetyl glycoprotein, unsaturated lipid, isoleucine, and VLDL/LDL are higher (p < 0.05) in the obese with hyperlipidemia. Therefore, they can be used as biomarkers to identify these two types of obesity.

Protein binding of 4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid to human serum albumin and their anti-proliferation on doxorubicin-sensitive and doxorubicin-resistant leukemia cells.

4-Hydroxybenzoic acids (4-HBA) and 4-hydroxy-3-methoxybenzoic acid (Vanillic acid, VA) have exhibited several pharmacological activities. Generally, the biological activities of compounds are highly involved in the interaction between protein and compounds in blood plasma. The objective was to investigate the interaction of 4-HBA or VA with human serum albumin (HSA) and their anti-proliferation properties on doxorubicin-sensitive K562 and doxorubicin-resistant K562/Dox leukemia cells. The protein binding of 4-HBA or VA to HSA was investigated using fluorescence quenching at temperatures of 298 and 310 Kelvin (K) under the pH of 6.0, 7.4, and 8.0 conditions. The effect of 4-HBA and VA on anti-proliferation was also studied on doxorubicin-sensitive K562 and doxorubicin-resistant K562/Dox leukemia cells using resazurin assay. The results showed that 4-HBA and VA could interact with HSA. The fluorescence quenching process in HSA-4-HBA system might be attributed to static quenching mechanism. In contrast, a dynamic quenching mechanism might be mainly involved in the fluorescence quenching process in the HSA-VA system. Thermodynamic data suggested that the spontaneous interaction between HSA and 4-HBA or VA had occurred in the system and it also indicated that hydrogen bonds and Van der Waals forces contributed to the binding of HSA to 4-HBA or VA. In addition, 4-HBA and VA decreased K562 and K562/Dox cells viability in a dose- and time-dependence manner. In conclusions, the 4-HBA and VA could interact with HSA. In addition, the 4-HBA and VA decreased in cell viability for both doxorubicin-sensitive K562 and doxorubicin-resistant K562/Dox leukemia cells in a dose- and time-dependence manner. Therefore, these current studies could provide useful information about the nature of 4-HBA or VA binding to protein HSA and their anticancer activities in both of these types of leukemia cells. The cell death mechanisms should be investigated through future study.

Different responses of normal cells (red blood cells) and cancer cells (K562 and K562/Dox cells) to low-dose 137Cs gamma-rays.

High-dose radiation is deleterious to cells or tissues. However, the health risks of exposure to low-dose radiation remain unclear. The present study aimed to investigate the biological responses of low-dose gamma-ray in vitro exposure to normal red blood cells (RBCs) and erythroleukemia (K562 and K562/Dox) cancer cells. Cells were given a low dose of 0.03, 0.05 and 0.1 mGy of 137Cs gamma-rays (at a dose rate of 0.001 Gy/min) under in vitro conditions. Cells exposed to 0 Gy served as controls. Hemolysis and reactive oxygen species (ROS) were measured in exposed RBCs following exposure to low-dose gamma-rays. In addition, complete blood count (CBC) parameters were determined in irradiated whole blood. For irradiated K562 and K562/Dox cancer cells, ROS and mitochondrial activity were measured at 0, 30, 60 and 120 post-irradiation times. The results showed no change in the percentage of ROS and hemolysis in irradiated RBCs. The data indicated no perturbation in the CBC parameters in irradiated whole blood. By contrast, statistically significant dose-dependent increases in the percentage of ROS and decreases in the mitochondrial activity in the K562 and K562/Dox cancer cells were observed from 0 min up to 120 min post-irradiation. These findings concluded that there were differences in biological responses in normal cells (RBCs) and cancer cells (K562 and K562/Dox) to low-dose gamma-rays when cells were irradiated under in vitro conditions.

Effects of gadolinium-based magnetic resonance imaging contrast media on red blood cells and K562 cancer cells.

BACKGROUND: Gadolinium-based contrast media (GBCM) are commonly used in diagnostic magnetic resonance imaging (MRI) in clinical applications. The objective of this study is to evaluate the antioxidant properties and effects on red blood cells (RBCs) and K562 cancer cells of three GBCMs (i.e.; gadoterate meglumine, gadopentetate dimeglumine, and gadobenate dimeglumine) inin vitro levels. METHODS: For determiningin vitro antioxidant properties, the di (phenyl)-(2,4,6-trinitrophenyl) iminoazanium (DPPH) and ferric reducing ability of plasma (FRAP) assay were used. For determining effect on red blood cells, hemolysis, morphology and reactive oxygen species (ROS) were used. For determining effect on K562 cancer cells, cytotoxicity and ROS were used. The GBCM -exposed cells were compared to corresponding non-exposed control groups at various harvest times. RESULTS: The results show no changes occurring in the DPPH data. However, there were significant increases based on FRAP data in three GBCMs compared to the corresponding control at all concentrations. The ROS, morphology, and percentage of hemolysis in red blood cells indicated that no change had occurred in three GBCMs-exposed red blood cells compared to the corresponding non-exposed control groups at all harvest times. The percentage of cell viability in K562 cancer cells showed decreases in gadoterate meglumine- and gadobenate dimeglumine- in a concentration dependent manner, but did not show same in gadopentetate dimeglumine-exposed K562 cancer cells. The percentage of ROS in K562 cancer cells indicated that no change in three GBCMs-exposed cells had occurred when compared to the corresponding non-exposed control groups at all harvest times. CONCLUSION: These findings suggests thatin vitro antioxidant properties exhibited by those three GBCMs depends on their concentration and species of radical in testing assay. There were no toxic effects from those GBCMs when red blood cells were exposed in an in vitro condition. In addition, some of those GBCMs could induce cell death in cancer cells.

EVALUATION OF THE BONE MINERAL DENSITY OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) VIA DUAL-ENERGY X-RAY IMAGING OF TAILS.

Musculoskeletal problems are one of the top five causes of disease in elephants. However, recent blood chemistry analysis is the only routine protocol for bone mineral status evaluation, with no assessment method currently available for the direct measurement of elephant bone mineral density (BMD). This work applied the the dual-energy X-ray technique (DXA) technique for bone density assessment. The elephant's tail was chosen for the analysis to avoid the radiation harm. Twelve live Asian elephants (Elephas maximus) comprising eight males and four females with ages in the range of 4-77 yr were investigated. The BMD was calculated based on radiographic images acquired using the DXA technique carried out at 40 kVp 2 mAs and 50 kVp 2 mAs. Blood serum analysis of total calcium (Ca), phosphorus (Phos) and alkaline phosphatase (ALP) content was conducted in parallel with the physical examination to correlate age and BMD. Analyses produced an overall mean BMD value in the range of 0.54-1.39 g/cm2, with that of the males higher than that of the females. The BMD was found to be negatively correlated with age, Ca, and Phos, but not with ALP. In summary, the BMD analysis of an elephant's tail might be used with blood serum Ca and Phos to predict the overall bone mineral status of the animal.

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators.

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and studied. The new compounds were designed based on the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake assay on the doxorubicin-resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the combination of aromatic moieties. Docking results obtained by an in silico study supported the data obtained by the biological tests and a study devoted to establish the chemical stability in phosphate buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their apparent permeability and ATP consumption on other cell lines suggested that the compounds can behave as unambiguous or not transported substrates. The activity of these the compounds on the transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the BCRP overexpressing cells; while only four molecules showed to be effective on MRP1 overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we have identified a new very powerful series of compounds which represent interesting leads for the development of new potent and efficacious P-gp-dependent MDR modulators.

The Effects of Iodinated Radiographic Contrast Media on Multidrug-resistant K562/Dox Cells: Mitochondria Impairment and P-glycoprotein Inhibition.

Iodinated radiographic contrast media is used in cancer radiography for cancer diagnosis. The aim of this present study was to examine five iodinated radiographic contrast media (IRCM) (i.e., iohexol, iopamidol, iobitridol, ioxaglate, and iodixanol) in terms of their cytotoxicity, mitochondria membrane potential (ΔΨm), and P-glycoprotein function in multidrug resistant K562/Dox cancer cells and corresponding sensitive cancer cells. The cytotoxicity was determined by colorimetric resazurin reduction assay. The ΔΨm and P-glycoprotein function was measured using a noninvasive functional spectrofluorometry. Rhodamine B, fluorescence probe, was used to estimate ΔΨm. The kinetic of P-glycoprotein-mediated efflux pirarubicin was used to monitor P-glycoprotein function in multidrug resistant (MDR) cancer cells. The results showed that ioxaglate and iodixanol show similar efficacy in MDR cancer cells and for their corresponding sensitive cancer cells. Iopamidol, iohexol, and iobitridol showed higher efficacy in MDR cancer cells than for the corresponding sensitive cancer cells by approximately 2 fold. The results also showed no significant change in the |ΔΨm| values in treated K562 and K562/Dox cancer cells when compared to the non-treated K562 and K562/Dox cancer cells. However, there were notable changes detected for iobitridol and iodixanol at 50 mgI/mL. Similarly, the results showed significant differences in P-glycoprotein function of K562/Dox cancer cells after treatment with IRCM when compared to the non-treated K562/Dox cancer cells, with iohexol and iodixanol being the notable exceptions once again. In this present study, IRCM exhibited cytotoxicity on MDR cancer cells and their corresponding sensitive cancer cells. IRCM also showed potential as an anticancer agent in the future.

Design and synthesis of new potent N,N-bis(arylalkyl)piperazine derivatives as multidrug resistance (MDR) reversing agents.

A series of 1,4-substituted arylalkyl piperazine derivatives were synthesized and studied with the aim to obtain potent P-gp-dependent multidrug-resistant (MDR) reversers. The new compounds were designed on the basis of the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity. All new compounds were active in the pirarubicin uptake assay on the doxorubicin-resistant erythroleukemia K562 cells (K562/DOX). In particular, compounds bearing methoxy aromatic moieties showed fairly high reversal activities. The most potent compounds, 8, 9, 10 and 13, were further studied by evaluating their doxorubicin cytotoxicity enhancement (reversal fold, RF) and the inhibition of P-gp-mediated rhodamine-123 (Rhd 123) efflux on the K562/DOX cell line. The results of all pharmacological assays indicated that the combination of a basic piperazine scaffold with arylalkyl residues allowed us to obtain very interesting P-gp modulating compounds. Two long-lasting P-gp pump modulators (9 and 10) were identified; they were able to inhibit remarkably the P-gp substrate rhodamine-123 efflux on the resistant K562/DOX cell line after 60 min. Overall compound 9 appeared the most promising compound being a potent and long-lasting P-gp-dependent MDR modulator.

Late Effects of Low-Dose Radiation on the Bone Marrow, Lung, and Testis Collected From the Same Exposed BALB/cJ Mice.

We used 3 biological metrics highly relevant to health risks, that is, cell death, inflammation, and global DNA methylation, to determine the late effects of low doses (0.05 or 0.1 Gy) of 137Cs γ rays on the bone marrow, lung, and testis collected at 6 months post-irradiation from the same exposed BALB/cJ mouse. This integrative approach has not been used for such a purpose. Mice exposed to 0 or 1 Gy of radiation served as a sham or positive control group, respectively. The results could deliver information for better health risk assessment across tissues, including better scientific basis for radiation protection and clinical application. We found no changes in the levels of all studied biological metrics (except a significant increase in the levels of an anti-inflammatory cytokine, ie, interleukin 10) in tissues of 0.05-Gy exposed mice, when compared to those in sham controls. In contrast, significantly increased levels of cell death and inflammation, including a significant loss of global 5-hydroxymethylcytosine, were found in all tissues of the same mice exposed to 0.1 or 1.0 Gy. Our data demonstrated not only no harm but also hormesis in the 0.05-Gy exposed mice. However, the hormetic effect appears to be dependent on biological metrics and tissue.

Visualization of Inflammation at Early Stage of Lung Cancer in Xenografted Temporally Immunosuppression Rats by Ferrioxamine Magnetic Resonance Imaging.

Physiological responses such as chronic inflammation and angiogenesis could be used as biomarkers for early detection of cancer with noninvasive imaging modalities. The present study reports the application of magnetic resonance imaging instrument to image the binding of ferrioxamine with hemin that allows visualizing the chronic inflammation foci of lung tissue of immunocompromised rats xenografted using small cell lung carcinoma. A low concentration of ferrioxamine (0.05 ± 0.02 µM·kg-1 of rat weight) deposited on tissue outside the vasculature was found to diffuse across the capillary walls to the interstitial space and inflammation foci, which provided a clear enhancement of T1-weighted gradient-echo sequence images. Ferrioxamine imaging allowed the determination of inflammatory sites and their localization in 3D fat-suppressed maximum intensity projections. The smallest dimension of foci that can be clearly determined is about 0.1 mm3. In concomitant to the in vivo imaging, analysis of histological tissue section showed the development of inflammatory sites. This study provides evidence that medical imaging instrument such as MRI scanner allows researchers to correlate images taken with MRI with those using high-resolution microscopy. Moreover, ferrioxamine is a useful molecular probe for determining chronic inflammation particularly at the very early stages of cancer.

Response to Baverstock, K. Comments on Rithidech, K.N.; et al. Lack of Genomic Instability in Bone Marrow Cells of SCID Mice Exposed Whole-Body to Low-Dose Radiation. Int. J. Environ. Res. Public Health 2013, 10, 1356-1377.

We thank Dr. Baverstock [1] for his interest in reading our article and his time in writing his comments for our work [2]. We, however, respectfully disagree with his statement that we made "two category errors" associated with the assessment of the occurrence of "genomic instability" by determining the frequencies of delayed- or late-occurring chromosomal damage. Our disagreement is based upon the well-known fact that radiation-induced genomic instability (or delayed/late-occurring damage) can be manifested in many ways. These include late-occurring chromosomal damage, or mutations, or gene expression, or gene amplifications, or transformation, or microsatellite instability, or cell killing [3-9]. Such phenomena have been detected many cell generations after irradiation. We agree that genomic instability may well be the consequence of epigenetic changes. Another mechanism mentioned by Dr. Bavertock as being probably unlikely is the reversibility of damage. This potential may not be discarded off-hand, as Dr. Baverstock prefers to do. There is much reproducible evidence of adaptive protection that depending on absorbed dose precisely may reverse early damage, and damage appearing late may be due to some form of residual damage letting the cell become genetically unstable. In other words, the argument by Dr. Baverstock regarding upward or downward causation appears to be rather speculative and far from being settled.

Lack of genomic instability in bone marrow cells of SCID mice exposed whole-body to low-dose radiation.

It is clear that high-dose radiation is harmful. However, despite extensive research, assessment of potential health-risks associated with exposure to low-dose radiation (at doses below or equal to 0.1 Gy) is still challenging. Recently, we reported that 0.05 Gy of 137Cs gamma rays (the existing limit for radiation-exposure in the workplace) was incapable of inducing significant in vivo genomic instability (measured by the presence of late-occurring chromosomal damage at 6 months post-irradiation) in bone marrow (BM) cells of two mouse strains, one with constitutively high and one with intermediate levels of the repair enzyme DNA-dependent protein-kinase catalytic-subunit (DNA-PKcs). In this study, we present evidence for a lack of genomic instability in BM cells of the severely combined-immunodeficiency (SCID/J) mouse (which has an extremely low-level of DNA-PKcs activity) exposed whole-body to low-dose radiation (0.05 Gy). Together with our previous report, the data indicate that low-dose radiation (0.05 Gy) is incapable of inducing genomic instability in vivo (regardless of the levels of DNA-PKcs activity of the exposed mice), yet higher doses of radiation (0.1 and 1 Gy) do induce genomic instability in mice with intermediate and extremely low-levels of DNA-PKcs activity (indicating an important role of DNA-PKcs in DNA repair).

No Evidence for the In Vivo Induction of Genomic Instability by Low Doses of CS Gamma Rays in Bone Marrow Cells of BALB/CJ and C57BL/6J Mice.

In spite of extensive research, assessment of potential health risks associated with exposure to low-dose (≤ 0.1 Gy) radiation is still challenging. We evaluated the in vivo induction of genomic instability, expressed as late-occurring chromosome aberrations, in bone-marrow cells of two strains of mouse with different genetic background, i.e. the radiosensitive BALB/cJ and the radioresistant C57BL/6J strains following a whole-body exposure to varying doses of (137)Cs gamma rays (0, 0.05, 0.1, and 1.0 Gy). A total of five mice per dose per strain were sacrificed at various times post-irradiation up to 6 months for sample collections. Three-color fluorescence in situ hybridization for mouse chromosomes 1, 2, and 3 was used for the analysis of stable-aberrations in metaphase-cells. All other visible gross structural-abnormalities involving non-painted-chromosomes were also evaluated on the same metaphase-cells used for scoring the stable-aberrations of painted-chromosomes. Our new data demonstrated in bone-marrow cells from both strains that low doses of low LET-radiation (as low as 0.05 Gy) are incapable of inducing genomic instability but are capable of reducing specific aberration-types below the spontaneous rate with time post-irradiation. However, the results showed the induction of genomic instability by 1.0 Gy of (137)Cs gamma rays in the radiosensitive strain only.