Hae Dong Woo

Effect of Bupleuri Radix Extracts on the Toxicity of 5‐Fluorouracil in HepG2 Hepatoma Cells and Normal Human Lymphocytes

Despite the excellent chemotherapeutic effect of 5-fluorouracil, its cytotoxicity and genotoxicity in normal cells remain a major problem. We sought to assess whether Bupleuri Radix extract enhances 5-fluorouracil-induced cytotoxicity in HepG2 hepatoma cells, while protecting normal blood lymphocytes. Bupleuri Radix, used for treatment of liver disease in oriental medicine, possesses antitumour properties; it induces apoptosis through cell arrest in tumour cells, but does not affect normal lymphocytes. In this study, we evaluated the protective and enhancing effects of Bupleuri Radix on 5-fluorouracil-induced cytotoxicity in HepG2 cells and normal lymphocytes. Treatment with Bupleuri Radix increased the micronuclei frequency and DNA damage, resulting from 5-fluorouracil treatment. However, when human lymphocytes were cotreated with Bupleuri Radix and 5-fluorouracil, the frequency of 5-fluorouracil-induced micronuclei decreased. Although the extent of 5-fluorouracil-induced DNA damage, determined by single-cell gel electrophoresis, increased after treating HepG2 cells with Bupleuri Radix, it decreased in normal lymphocytes. When cells were treated with 20 microM 5-fluorouracil and 200 microg/ml Bupleuri Radix simultaneously, Bax protein increased in HepG2 cells at 24 hr; however, p21 and p53 proteins were up-regulated in normal human lymphocytes. Cotreatment with 200 microg/ml Bupleuri Radix and 20 microM 5-fluorouracil resulted in cell arrest at the late G(1)/early S phase in HepG2 cells (55.80 +/- 0.19%) and normal lymphocytes (97.19 +/- 0.27%). In addition, Bupleuri Radix and 5-fluorouracil treatment increased mitochondria membrane potential collapse only in HepG2 cells (19.02%), while it was not changed in lymphocytes. In conclusion, our findings suggest that Bupleuri Radix may be effective as a therapeutic agent to treat hepatomas.

Micronucleus-centromere assay and DNA repair gene polymorphism in lymphocytes of industrial radiographers

The micronucleus-centromere assay using a pan-centromeric probe was used to assess chromosomal damage in lymphocytes of 47 industrial radiographers occupationally exposed to low dose ionizing radiation and 47 controls. The influence of genotype of DNA repair genes (XRCC1(399), XRCC3(241) and XPD(751)) on micronuclei (MN) frequency was also investigated. Centromere negative micronuclei (MNC-) frequency was significantly higher in radiographers than in controls, whereas similar centromere positive micronuclei (MNC+) frequency was observed in both groups. Poisson regression analyses revealed that the MNC- frequency was significantly associated with radiation occupational exposure and with cumulative-radiation doses in radiographers, after adjusting for confounding variables such as age, smoking, alcohol intake and genotypes. Compared to homozygous wild-type subjects, MNC- frequency in radiographers with variant XRCC3 genotype was significantly higher using univariate analysis. There were no differences in MNC- or MNC+ frequencies by genotype in controls. In conclusion, scoring of MNC- is a useful cytogenetic biomonitoring method for radiographers. Polymorphisms in XRCC3 might contribute to the increased genetic damage in individuals occupationally exposed to chronic ionizing radiation.

Risk Assessment of Hydroquinone: Differential Responses of Cell Growth and Lethality Correlated to Hydroquinone Concentration

Hydroquinone (HQ) is a major metabolite of benzene and has been used as an antioxidant, a stabilizer, a photographic reducer, and an ingredient in skin lighteners. In this study, the effects of low (5 μM) and high (50 μM) concentrations of HQ were investigated on cell growth and lethality in Jurkat cells. Intracellular reactive oxygen species (ROS) levels were increased with both HQ concentrations. Fifty micromolar HQ markedly increased phosphorylation of ERK and activation of caspase-9/-3, followed by PARP cleavage. The addition of ERK inhibitor PD98059 or N-acetylcysteine (NAC) abolished HQ-induced apoptosis. Five micromolar HQ activated ERK protein, but not JNK or p38. However, S-phase recruitment was decreased by preincubation with NAC, but not PD98059. Thus, high levels of ROS contributed to HQ-induced apoptosis via ERK signaling and the caspase pathway, whereas low quantities of ROS resulted in S-phase recruitment in the cell-cycle distribution.

Micronucleus-centromere assay in workers occupationally exposed to low level of benzene

Benzene is a well-known carcinogen that induces chromosomal instability, including chromosome aberration and aneuploidy. In order to assess aneugenic effect of low-level benzene, micronucleus-centromere assay using specific probes for chromosomes 7 and 9 was performed in workers occupationally exposed to low-dose benzene at a petroleum refinery. A micronucleus-centromere assay using a pan-centromeric probe was also performed to determine the origin of benzene-induced micronucleus (MN). Frequency of aneuploidy of chromosomes 7 and 9 was significantly higher among workers compared to the unexposed control group. Poisson regression analysis revealed that aneuploidy frequency of chromosome 7 or 9 was significantly associatedwithbenzenelevelafteradjustingforconfoundingvariablessuchasage,smoking,alcoholintake,andduration of work (p ¼ .042). Additionally, frequencies of MN and centromere-negative micronuclei (MNC–) were significantly higher in benzene-exposed workers compared to controls, while frequency of centromere-positive micronuclei (MNCþ) was similar in both groups. In conclusion, aneuploidy of chromosomes 7 and 9 could be a useful biomarker to assess the effect of low-level benzene exposure, and benzene-induced MN originates from chromosome breaks rather than chromosome non-disjunction.Benzene is a well-known carcinogen that induces chromosomal instability, including chromosome aberration and aneuploidy. In order to assess aneugenic effect of low-level benzene, micronucleus-centromere assay using specific probes for chromosomes 7 and 9 was performed in workers occupationally exposed to low-dose benzene at a petroleum refinery. A micronucleus-centromere assay using a pan-centromeric probe was also performed to determine the origin of benzene-induced micronucleus (MN). Frequency of aneuploidy of chromosomes 7 and 9 was significantly higher among workers compared to the unexposed control group. Poisson regression analysis revealed that aneuploidy frequency of chromosome 7 or 9 was significantly associated with benzene level after adjusting for confounding variables such as age, smoking, alcohol intake, and duration of work (p = .042). Additionally, frequencies of MN and centromere-negative micronuclei (MNC—) were significantly higher in benzene-exposed workers compared to controls, while frequency of centromere-positive micronuclei (MNC+) was similar in both groups. In conclusion, aneuploidy of chromosomes 7 and 9 could be a useful biomarker to assess the effect of low-level benzene exposure, and benzene-induced MN originates from chromosome breaks rather than chromosome non-disjunction.

Quercetin prevents necrotic cell death induced by co-exposure to benzo(a)pyrene and UVA radiation.

The phototoxicity of low-energy ultraviolet radiation, such as UVA, can be enhanced by the presence of photosensitizing agents. Hence, co-exposure of cells to benzo[a]pyrene (BaP), a widespread environmental carcinogen and photosensitizing agent, and UVA may synergistically induce DNA damage. In this study, exposure of cells to various concentrations of BaP for 1h followed by UVA irradiation (2J/cm(2)) increased DNA damage and decreased cell viability. Expression of apoptosis-related proteins (caspase-9, caspase-3, PARP, and Bax) and hypodiploid DNA content (sub-G(1)) were not changed. LDH release into the culture medium increased in a dose-dependent manner with BaP under UVA irradiation, suggesting that cell death due to BaP/UVA co-treatment occurred via necrosis. Intracellular reactive oxygen species (ROS) levels were increased significantly in the co-exposed cells, and treatment with the polyphenol quercetin, but not with sodium azide or N-acetylcysteine, decreased ROS levels and increased cell viability in BaP/UVA-treated cells. In conclusion, UVA irradiation combined with BaP synergistically promoted necrosis of A549 cells by increasing intracellular ROS levels, and quercetin prevented BaP-enhanced phototoxicity due to UVA irradiation.

The Association of LINE-1 Hypomethylation with Age and Centromere Positive Micronuclei in Human Lymphocytes

Global hypomethylation in white blood cell (WBC) DNA has recently been proposed as a potential biomarker for determining cancer risk through genomic instability. However, the amplitude of the changes associated with age and the impacts of environmental factors on DNA methylation are unclear. In this study, we investigated the association of genomic hypomethylation with age, cigarette use, drinking status and the presence of centromere positive micronuclei (MNC+)—a biomarker for age-dependent genomic instability. Genomic hypomethylation of the repetitive element LINE-1 was measured in WBC DNA from 32 healthy male volunteers using the pyrosequencing assay. We also measured MNC+ with the micronucleus-centromere assay using a pan-centromeric probe. Possibly due to the small sample size and resulting low statistical power, smoking and drinking status had no significant effect on LINE-1 hypomethylation or the occurrence of MNC+. Consequently, we did not include them in further analyses. In contrast, LINE-1 hypomethylation and age significantly predicted MNC+; therefore, we examined whether LINE-1 hypomethylation plays a role in MNC+ formation by age, since genomic hypomethylation is associated with genomic instability. However, LINE-1 hypomethylation did not significantly mediate the effect of age on MNC+. Our data indicate that the repetitive element LINE-1 is demethylated with age and increasing MNC+ frequency, but additional studies are needed to fully understand the relation between genomic DNA hypomethylation, age and genomic instability.

Protective Effect of Onion Extract on Bleomycin-Induced Cytotoxicity and Genotoxicity in Human Lymphocytes.

Following one of the world’s largest nuclear accidents, occured at Fukushima, Japan in 2011, a significant scientific effort has focused on minimizing the potential adverse health effects due to radiation exposure. The use of natural dietary antioxidants to reduce the risk of radiation-induced oxidative DNA damage is a simple strategy for minimizing radiation-related cancer rates and improving overall health. The onion is among the richest sources of dietary flavonoids and is an important food for increasing their overall intake. Therefore, we examined the effect of an onion extract on cyto- and geno-toxicity in human lymphocytes treated with bleomycin (BLM), a radiomimetic agent. In addition, we measured the frequency of micronuclei (MN) and DNA damage following treatment with BLM using a cytokinesis-blocked micronucleus assay and a single cell gel electrophoresis assay. We observed a significant increase in cell viability in lymphocytes treated with onion extract then exposed to BLM compared to cells treated with BLM alone. The frequency of BLM induced MN and DNA damage increased in a dose-dependent manner; however, when lymphocytes were pretreated with onion extract (10 and 20 μL/mL), the frequency of BLM-induced MN was decreased at all doses of BLM and DNA damage was decreased at 3 μg/mL of BLM. These results suggest that onion extract may have protective effects against BLM-induced cyto- and genotoxicity in human lymphocytes.

Delayed Numerical Chromosome Aberrations in Human Fibroblasts by Low Dose of Radiation.

Radiation-induced genomic instability refers to a type of damage transmitted over many generations following irradiation. This delayed impact of radiation exposure may pose a high risk to human health and increases concern over the dose limit of radiation exposure for both the public and radiation workers. Therefore, the development of additional biomarkers is still needed for the detection of delayed responses following low doses of radiation exposure. In this study, we examined the effect of X-irradiation on delayed induction of numerical chromosomal aberrations in normal human fibroblasts irradiated with 20, 50 and 100 cGy of X-rays using the micronucleus-centromere assay. Frequencies of centromere negative- and positive-micronuclei, and aneuploidy of chromosome 1 and 4 were analyzed in the surviving cells at 28, 88 and 240 h after X-irradiation. X-irradiation increased the frequency of micronuclei (MN) in a dose-dependent manner in the cells at all measured time-points, but no significant differences in MN frequency among cell passages were observed. Aneuploid frequency of chromosomes 1 and 4 increased with radiation doses, and a significantly higher frequency of aneuploidy was observed in the surviving cells analyzed at 240 h compared to 28 h. These results indicate that low-dose of X-irradiation can induce delayed aneuploidy of chromosomes 1 and 4 in normal fibroblasts.

Cytotoxic Effects on HL-60 Cells of Myosin Light Chain Kinase Inhibitor ML-7 Alone and in Combination with Flavonoids

Uncontrolled cell growth and increased cell proliferation are major features of cancer that are dependent on the stable structure and dynamics of the cytoskeleton. Since stable cytoskeleton structure and dynamics are partly regulated by myosin light chain kinase (MLCK), many current studies focused on MLCK inhibition as a chemotherapeutic target. As a potent and selective MLCK inhibitor, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine hydrochloride] is a promising candidate for an anticancer agent, which would induce apoptosis as well as prevents invasion and metastasis in certain types of cancer cells. This study assessed cytotoxic effects of ML-7 against HL-60 cells and therapeutic efficacy of ML-7 as a potential antileukemia agent. Trypan-blue exclusion assays showed dose- and time- dependent decreases in ML-7 treated HL-60 cells (p < 0.05). Comet assays revealed a significant increase in DNA damage in HL-60 cells after treatment with 40 µM ML-7 for 2 h. Sub-G1 fractions, analyzed by flow cytometry increased in a dose-dependent manner, suggesting that ML-7 can induce apoptotic cell death in HL-60 cells. ML-7 was selectively cytotoxic towards HL-60 cells; not affecting normal human lymphocytes. That selective effect makes it a promising potential anti-leukemia agent. In addition, anticancer efficacy of ML-7 in combination with flavonoids (genistein or quercetin) or anticancer drugs (cisplatin or AraC) against HL-60 cells was assessed. Combination of ML-7 with flavonoids increased the anticancer effect of ML-7 to a greater extent than combination with the anticancer drugs. This implies that ML-7 in combination with flavonoids could increase the efficacy of anticancer treatment, while avoiding side effects cansed by conventional anticancer drug-containing combination chemotherapy.