Biomedical subjects

Cheng-Hung Chuang

Publications and source records attributed to Cheng-Hung Chuang.

6 recordsLinked to original sources

PubMed2006

Synergistic DNA damage and lipid peroxidation in cultured human white blood cells exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and ultraviolet A.

4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen in cigarette smoke, while ultraviolet (UV) irradiation from sunlight is a major factor for causing skin aging and skin cancer. However, little is known about the effects of the interaction between NNK and UV light on the induction of DNA damage and oxidative stress. In this study, we incubated human white blood cells (WBCs) with NNK, followed by irradiating the cells with ultraviolet A (UVA) (320-380 nm), and we measured DNA strand breaks (by the Comet or single-cell gel electrophoresis assay), lipid peroxidation (as thiobarbituric acid-reactive substances, TABRS), and the levels of intracellular reactive oxygen species (ROS). We found that preincubation with 1.0 mM NNK, followed by UVA irradiation (7.6 kJ/m2) synergistically increased DNA damage, lipid peroxidation, and the level of intracellular ROS in WBCs, while NNK or UVA alone had little or no effect. Electron spin resonance spectroscopic analyses showed that NNK plus UVA enhanced the UVA-induced generation of singlet oxygen but not hydroxyl radicals. In addition to ROS, bioactivation of NNK by cytochromes P450 (CYP) to form reactive NNK intermediates may also be involved in the synergistic damage to WBCs by NNK plus UVA. This is evidenced by the synergistic increase in N7-methylguanine (7-mGua), a major DNA adduct produced by NNK. Overall, the present study demonstrates that exposure of WBCs to both NNK and UVA synergistically increases DNA damage and lipid peroxidation and that such effects involve enhanced generation of ROS, especially singlet oxygen, and activation of NNK to 7-mGua by CYP. The results imply that NNK is a phototoxic agent.

Cell Survival↗

PubMed2005

Effect of dietary vitamin E and selenium on DNA damage in fresh and frozen tissues.

To determine whether deficiencies of dietary vitamin E and Se can elevate background DNA damage, rats were fed diets deficient in or supplemented with vitamin E (30 and 200 mg/kg diet) and Se (0.2 mg/kg diet) for 8 weeks. DNA damage was measured using the Comet (single-cell electrophoresis) assay and 8-oxodeoxyguanosine (8-oxo-dG) in liver, kidneys, and lymphocytes. We found that a deficiency of vitamin E and/or Se for 8 weeks did not significantly increase DNA damage in freshly isolated liver, kidneys, or lymphocytes. However, deficiency of vitamin E and/or Se for 8 weeks markedly increased DNA strand breaks in frozen kidney (-80 degrees C for 72 hours) and in lymphocytes incubated overnight at 37 degrees C, both of which were effectively prevented by supplementation of Se and vitamin E. However, vitamin E at 200 mg/kg did not afford more protection than it did at 30 mg/kg). Little or no significant increase in DNA damage was found in frozen livers. These results indicate that freezing or freeze-thawing of tissues may cause oxidative damage to DNA when the tissues are deficient in a major antioxidant, and that normal levels of vitamin E (30 mg/kg diet) and Se (0.2 mg/kg diet) are sufficient to prevent the damage. Thus, our results caution against the interpretation of DNA data obtained from frozen rat tissues or cells in animal studies with dietary vitamin E or Se deficiencies.

Animals↗

PubMed2005

Lycopene inhibits cell migration and invasion and upregulates Nm23-H1 in a highly invasive hepatocarcinoma, SK-Hep-1 cells.

The carotenoid lycopene has been associated with decreased risks of several types of cancer, such as prostate cancer and hepatoma. Tumor metastasis is the most important cause of cancer death. Although lycopene was shown to inhibit metastasis, the mechanism underlying this action is not well understood. Here, we tested the possibility that lycopene may inhibit cancer cell metastasis by upregulating the expression of nm23-H1, a metastasis suppressor gene, in SK-Hep-1 cells, a highly invasive hepatoma cell line, and we determined migration and invasion activities and the expression of nm23-H1 protein and mRNA. We showed that lycopene inhibited SK-Hep-1 migration and invasion in a bell-shaped manner, with the highest effect at 5 micromol/L (91 and 63% inhibition for migration and invasion, respectively; P < 0.05). At the same test level (10 micromol/L), lycopene was much more effective than beta-carotene in reducing cell invasion (by approximately 870%). In contrast to the effects on migration and invasion, lycopene enhanced nm23-H1 expression at both the protein and mRNA levels; the effects were also bell shaped, and at 5 micromol/L, lycopene enhanced nm23-H1 protein and mRNA expressions by 220 +/- 33 and 153 +/- 22% (P < 0.01), respectively. These bell-shaped effects of lycopene may be related to autoxidation of lycopene at elevated concentrations (> or =10 micromol/L). Significant correlations existed between nm23-H1 protein expression and migration (r2= 0.78, P < 0.001) and between nm23-H1 protein expression and invasion (r2= 0.84, P < 0.001) in lycopene-treated SK-Hep-1 cells. We conclude that lycopene has significant antimigration and anti-invasion activity, and that this effect is associated with its induction of nm23-H1 expression.

Anticarcinogenic Agents↗

PubMed2004

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay.

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.

Animals↗

PubMed2004

A downstream algorithm based on extended gradient vector flow field for object segmentation.

For object segmentation, traditional snake algorithms often require human interaction; region growing methods are considerably dependent on the selected homogeneity criterion and initial seeds; watershed algorithms, however, have the drawback of over segmentation. A new downstream algorithm based on a proposed extended gradient vector flow (E-GVF) field model is presented in this paper for multiobject segmentation. The proposed flow field, on one hand, diffuses and propagates gradients near object boundaries to provide an effective guiding force and, on the other hand, presents a higher resolution of direction than traditional GVF field. The downstream process starts with a set of seeds scored and selected by considering local gradient direction information around each pixel. This step is automatic and requires no human interaction, making our algorithm more suitable for practical applications. Experiments show that our algorithm is noise resistant and has the advantage of segmenting objects that are separated from the background, while ignoring the internal structures of them. We have tested the proposed algorithm with several realistic images (e.g., medical and complex background images) and gained good results.

Algorithms↗

PubMed2002

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues.

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 units/g liver at 37 degrees C for 20 min) for isolation of intact cells for subsequent comet assay We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation (-85 degrees C for 72 h). To minimize this damage, minced liver (1.0 g) and kidney (0.5 g) tissues were added to 20 ml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO (-85 degrees C for 72 h and 3 weeks), and to a slightly lesser extent with glycerol (72 h), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150units/g liver and 300units/g kidney for 10 min produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at - 85 degrees C for 72 h. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.

Animals↗