Steven B. Zhang

Antioxidant Properties of Quercetin

UNLABELLED Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

A basic fibroblast growth factor analog for protection and mitigation against acute radiation syndromes.

AbstractThe effects of fibroblast growth factors and their potential as broad-spectrum agents to treat and mitigate radiation injury have been studied extensively over the past two decades. This report shows that a peptide mimetic of basic fibroblast growth factor (FGF-P) protects and mitigates against acute radiation syndromes. FGF-P attenuates both sepsis and bleeding in a radiation-induced bone marrow syndrome model and reduces the severity of gastrointestinal and cutaneous syndromes; it should also mitigate combined injuries. FGF‐2 and FGF-P induce little or no deleterious inflammation or vascular leakage, which distinguishes them from most other growth factors, angiogenic factors, and cytokines. Although recombinant FGFs have proven safe in several ongoing clinical trials, they are expensive to synthesize, can only be produced in limited quantity, and have limited shelf life. FGF-P mimics the advantageous features of FGF‐2 without these disadvantages. This paper shows that FGF-P not only has the potential to be a potent yet safe broad-spectrum medical countermeasure that mitigates acute radiotoxicity but also holds promise for thermal burns, ischemic wound healing, tissue engineering, and stem-cell regeneration.Health Phys. 106(6):000-000; 2014

B1 Sequence-based real-time Quantitative PCR: A sensitive method for direct measurement of mouse plasma DNA levels after gamma irradiation

PURPOSE Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. METHODS AND MATERIALS Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ((137)Cs gamma-ray source at approximately 1.86 Gy/min; homogeneity: +/- 6.5%). RESULTS The correlation coefficient between DNA levels and the threshold cycle value (C(T)) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples. CONCLUSIONS A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

Synthesis and anticancer potential of novel xanthone derivatives with 3,6-substituted chains.

In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and 6.09μM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.

Delayed Effects of Radiation on Mitochondrial DNA in Radiation-Sensitive Organs

Since Balb/c mice are sensitive to acute hematopoietic syndrome and C57BL/6 to late fibrovascular toxicity, we measured the effect of total body irradiation (TBI) on mitochondrial DNA (mtDNA) copy number in these two strains. Six months after TBI, we extracted total genomic DNA, including nuclear DNA (nDNA) and mtDNA, from various tissues. The findings included: (1) TBI had no significant effect on body weight for Balb/c or C57BL/6 when compared to aged controls. (2) For different organs in control mice, the ratio of mtDNA to nDNA ranged from 0.33 to 11.53 in Balb/c and 0.34 to 13.28 in C57BL/6. (3) Ratios of mtDNA to nDNA were generally similar to or higher in C57BL/6 compared to Balb/c. (4) In C57BL/6, TBI induced an increase in mitochondrial copy number only in the brain (p < 0.05). (5) In Balb/c, radiation induced a dose-dependent increase of mtDNA in the liver (p < 0.05 and p < 0.01) and a decrease in skeletal/muscle tissue (p < 0.01). Our findings indicate that radiation influences mtDNA copy number differently in various tissues. This may explain differences in late radiation toxicity to those organs and differences in radiation tolerance between murine strains.

Triptolide mitigates radiation-induced pneumonitis via inhibition of alveolar macrophages and related inflammatory molecules

Ionizing radiation-induced pulmonary injury is a major limitation of radiotherapy for thoracic tumors. We have demonstrated that triptolide (TPL) could alleviate IR-induced pneumonia and pulmonary fibrosis. In this study, we explored the underlying mechanism by which TPL mitigates the effects of radiotoxicity. The results showed that: (1) Alveolar macrophages (AMs) were the primary inflammatory cells infiltrating irradiated lung tissues and were maintained at a high level for at least 17 days, which TPL could reduce by inhibiting of the production of macrophage inflammatory protein-2 (MIP-2) and its receptor CXCR2. (2) Stimulated by the co-cultured irradiated lung epithelium, AMs produced a panel of inflammative molecules (IMs), such as cytokines (TNF-α, IL-6, IL-1α, IL-1β) and chemokines (MIP-2, MCP-1, LIX). TPL-treated AMs could reduce the production of these IMs. Meanwhile, AMs isolated from irradiated lung tissue secreted significantly high levels of IMs, which could be dramatically reduced by TPL. (3) TPL suppressed the phagocytosis of AMs as well as ROS production. Our results indicate that TPL mitigates radiation-induced pulmonary inflammation through the inhibition of the infiltration, IM secretion, and phagocytosis of AMs.

Maternal Bias in Mouse Radiosensitivity: The Role of the Mitochondrial PTP

This study investigated, at the molecular level, mitochondrial responses to radiation. In three mouse strains, we found the following: (1) mitochondrial response to calcium stress was associated with a strains susceptibility to γ-radiation; (2) γ-radiation increased this calcium stress response in a dose-responsive manner; (3) the mitochondrial DNA (mtDNA) copy number in the liver of the radiosensitive mouse strain was significantly lower, as compared to that of the radioresistant strain; (4) adenine nucleotide translocase (ANT) mRNA copy numbers were significantly lower in the radiosensitive strain; (5) the F1 offspring (BC/C57M) of radiosensitive females mated with radioresistant males exhibited a significant difference in calcium stress response from that of the radiation-resistant strain, but the reverse cross did not exhibit this difference; and (6) only those mitochondria extracted from the livers of irradiated BC/C57M mice exhibited a heightened calcium stress response. We propose that a genetic change in ANT and a postirradiation change involving either mtDNA-encoded protein replacement or altered mtDNA association fit these data.

Mitochondrial DNA and functional investigations into the radiosensitivity of four mouse strains.

We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive), C57BL/6 (radioresistant), and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP) measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

Mitochondrial Genetic Abnormalities After Radiation Exposure

Because mitochondria are prone to oxidative stress, damage to their DNA might provide a record of radiation exposure. We measured the effect of gamma radiation on mitochondrial DNA (mtDNA) copy number and common deletion (mito-CD) mutations using Beas-2B and HFL-1 cells lines and C3H/HeJ mice exposed to total-body irradiation (TBI) and sub-TBI. DNA was extracted 5 days after cell irradiation or 12 months after animal exposure. We found that: (1) natural ratios of mtDNA/nDNA and mito-CD/mtDNA varied between cell lines; (2) mtDNA copy number decreased in Beas-2B and increased in HFL-1 following 2 Gy; (3) mito-CD in both cell lines increased after 2 Gy; (4) in aged mice, the natural ratios of mtDNA/nDNA varied from 0.723 to 8.146 in different tissues; (5) in kidney tissue, TBI and sub-TBI mildly increased mtDNA copy number but substantially increased mtDNA-CD; and (6) in liver tissue, TBI and sub-TBI induced a slight increase in mtDNA copy number and a larger increase in mtDNA-CD. These findings indicate that mtDNA copy number varies in time by cell type, but there is a substantial and sustained increase in mtDNA mutations that occurs to different degrees in different tissues and cells following irradiation.

The Murine Common Deletion: Mitochondrial DNA 3,860-bp Deletion after Irradiation

This study demonstrates that mice, similar to humans, have a common mitochondrial DNA deletion (3,860 bp) that encodes 5 transfer RNA genes and 5 polypeptide genes that is related to aging, tissue type and radiotoxicity. Our research indicates that the deletion ratio in the liver was significantly higher than in the brain and gut tissues of 8-month-old mice, as compared to 8-week-old mice. Our results also demonstrate that tissue type, oxidative metabolic capacity and radiosensitivity influence the 3,860-bp deletion level. Therefore, this 3,860-bp deletion content may serve as a biomarker of aging and oxidative damage in mice.