Yang Jee Kim

Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells.

A safe alternative to the viral system used in gene therapy is a nonviral gene delivery system. Although polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer are among the most promising gene-carrier candidates for efficient nonviral gene delivery, safety concerns regarding their toxicity remain. The aim of this study was to scrutinize the underlying mechanism of the cytotoxicity and genotoxicity of PEI (25 kDa) and PAMAM (G4). To our knowledge, this is the first study to explore the genotoxic effect of polymeric gene carriers. To evaluate cell death by PEI and PAMAM, we performed propidium-iodide staining and lactate-dehydrogenase release assays. The genotoxicity of the polymers was measured by comet assay and cytokinesis-block micronucleus assay. PEI- and PAMAM-treated groups induced both necrotic and apoptotic cell death. In the comet assay and micronuclei formation, significant increases in DNA damage were observed in both treatments. We conclude that PEI and PAMAM dendrimer can induce not only a relatively weak apoptotic and a strong necrotic effect, but also a moderate genotoxic effect.

Genotoxic effects of 3 T magnetic resonance imaging in cultured human lymphocytes.

The clinical and preclinical use of high-field intensity (HF, 3 T and above) magnetic resonance imaging (MRI) scanners have significantly increased in the past few years. However, potential health risks are implied in the MRI and especially HF MRI environment due to high-static magnetic fields, fast gradient magnetic fields, and strong radiofrequency electromagnetic fields. In this study, the genotoxic potential of 3 T clinical MRI scans in cultured human lymphocytes in vitro was investigated by analyzing chromosome aberrations (CA), micronuclei (MN), and single-cell gel electrophoresis. Human lymphocytes were exposed to electromagnetic fields generated during MRI scanning (clinical routine brain examination protocols: three-channel head coil) for 22, 45, 67, and 89 min. We observed a significant increase in the frequency of single-strand DNA breaks following exposure to a 3 T MRI. In addition, the frequency of both CAs and MN in exposed cells increased in a time-dependent manner. The frequencies of MN in lymphocytes exposed to complex electromagnetic fields for 0, 22, 45, 67, and 89 min were 9.67, 11.67, 14.67, 18.00, and 20.33 per 1000 cells, respectively. Similarly, the frequencies of CAs in lymphocytes exposed for 0, 45, 67, and 89 min were 1.33, 2.33, 3.67, and 4.67 per 200 cells, respectively. These results suggest that exposure to 3 T MRI induces genotoxic effects in human lymphocytes.

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.

Association of the NQO1, MPO, and XRCC1 polymorphisms and chromosome damage among workers at a petroleum refinery.

Benzene is a leukemogen, and exposure to benzene is an occupational hazard in the petroleum refining industries. The effects of genetic polymorphisms in the NQO1 (rs1800566), MPO (rs2333227), and XRCC1 (rs25487) genes on benzene-induced chromosome abnormalities were assessed in 108 benzene-exposed workers and 33 office workers. The mean benzene exposure for exposed workers was 0.51 ppm for full-shift workers, and the time-weighted average ranged from 0.004 to 4.25 ppm. The frequencies of micronuclei (MN) and chromosome aberrations (CA) were significantly higher in workers exposed to benzene than unexposed controls. Exposed workers with the T/T genotype for NQO1 showed significant 1.9-fold (95% CI = 1.5–2.3) and 2.6-fold (95% CI = 1.7–3.9) increases in MN and CA frequencies, respectively, compared to controls with C/C and C/T genotypes, after adjusting for age, smoking status, and alcohol intake. Among exposed workers, subjects with the combination of MPO G/G and XRCC1 Arg/Gln or Gln/Gln showed a significantly higher CA frequency compared to those with the combination of MPO G/A or A/A and XRCC1 Arg/Arg genotypes. These results indicated that the genotoxicity induced by a chronic benzene exposure is modulated by genes involved in both DNA repair and benzene metabolic pathways.

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.

Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells.

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely used chemotherapeutic drugs, but its effectiveness is limited by tumor cell resistance and the severe side effects it causes. One strategy for overcoming this problem is the concomitant use of natural dietary compounds as therapeutic agents. Benzyl isothiocyanate (BITC) is a promising chemopreventive agent found in cruciferous vegetables and papaya fruits. The aim of this study was to investigate the effects of BITC on cisplatin-induced cytotoxicity in human promyelocytic leukemia cells and normal human lymphocytes. The combined treatment of HL-60 cells with BITC followed by cisplatin (BITC/cisplatin) caused a significant decrease in cell viability. BITC also increased apoptotic cell death compared to cisplatin treatment alone. In normal human lymphocytes, BITC did not enhance the cytotoxic effects of cisplatin. Cellular exposure to BITC/cisplatin increased reactive oxygen species (ROS) generation but decreased the total glutathione (GSH) level in HL-60 cells. Pretreatment of HL-60 cells with N-acetylcysteine or glutathione monoethyl ester effectively decreased BITC/cisplatin-induced cell death. The addition of the extracellular signal-regulated kinase (ERK) inhibitor PD98059 abolished BITC/cisplatin-induced apoptosis. Taken together, our results suggest that BITC enhances cisplatin-induced cytotoxicity through the generation of ROS, depletion of GSH, and ERK signaling in HL-60 cells.

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.

Determination of chromosome aberrations in workers in a petroleum refining factory.

Human exposure to benzene is derived occupationally from the petrochemical and petroleum refining industries. This study was performed to determine whether the frequencies of chromosome aberrations in workers exposed to low levels of benzene in a petroleum refining factory were elevated compared to an unexposed control group. The study population was comprised of 178 exposed workers and 36 unexposed workers. The frequencies of chromatid deletions and total chromosome aberrations in workers exposed to benzene were significantly higher compared to the unexposed control group. The frequency of total chromosome aberration was 4.20 per 500 metaphase cells in the exposed workers, whereas the frequency was 2.24 per 500 metaphase cells in the unexposed control group. The frequencies of total chromosome aberrations were significantly associated with benzene concentration after adjusting for confounding variables such as age, smoking status, and alcohol intake. The frequencies of chromosome aberrations were significantly increased in workers with low white blood cell counts (below 4000 cells/mm3) compared to those with high white blood cell counts (above 4000 cells/mm3). A reduced white blood cell count (below 4000/mm3) is suggestive of chronic exposure to benzene. In conclusion chronic benzene exposure and preclinical signs, such as reduced white blood cell counts, may be monitored by chromosome aberrations analysis.

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.