Young Joon Lee

Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes.

Titanium dioxide nanoparticles (nano‐TiO2) are widely used as a photocatalyst in air and water remediation. These nanoparticles are known to induce toxicity; however, their cytotoxic mechanism is not fully understood. In this study, we investigated the underlying mechanism of nano‐TiO2‐induced cytotoxicity in peripheral blood lymphocytes. We examined the genotoxic effects of nano‐TiO2 in lymphocytes using alkaline single‐cell gel electrophoresis (Comet) and cytokinesis‐block micronucleus (CBMN) assays. Lymphocytes treated with nano‐TiO2 showed significantly increased micronucleus formation and DNA breakage. Western‐blot analysis to identify proteins involved in the p53‐mediated response to DNA damage revealed the accumulation of p53 and activation of DNA damage checkpoint kinases in nano‐TiO2‐treated lymphocytes. However, p21 and bax, downstream targets of p53, were not affected, indicating that nano‐TiO2 does not stimulate transactivational activity of p53. The generation of reactive oxygen species (ROS) in nano‐TiO2‐treated cells was also observed, andN‐acetylcysteine (NAC) supplementation inhibited the level of nano‐TiO2‐induced DNA damage. Given that ROS‐induced DNA damage leads to p53 activation in the DNA damage response, our results suggest that nano‐TiO2 induces ROS generation in lymphocytes, thereby activating p53‐mediated DNA damage checkpoint signals. Environ. Mol. Mutagen., 2008.

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.

Cytotoxicity and genotoxicity of titanium dioxide nanoparticles in UVA-irradiated normal peripheral blood lymphocytes

The phototoxicity of ultraviolet A irradiation (UVA) can be enhanced by photosensitizing agents, such as titanium dioxide nanoparticles (100u2009nm in diameter, “normal-TiO2”). Nano-TiO2 treatment in the absence of UVA caused a slight decrease in cell viability, but in the presence of UVA, it caused a significant decrease in cell viability. In the presence of UVA, nano-TiO2 also significantly increased the percentage of the cell population in the sub-G1 phase, induced activation of the proapoptotic proteins, caspase-9, caspase-3, and poly(ADP)ribose polymerase, significantly increased the production of reactive oxygen species (ROS), and induced the loss of the mitochondrial membrane potential (MMP), suggesting that UVA and nano-TiO2 synergistically promoted apoptosis via a mitochondrial pathway. In the presence of UVA, but not in its absence, nano-TiO2 treatment also caused a significant increase in DNA damage. Normal-TiO2 used at the same concentrations did not cause DNA damage, induce ROS generation, trigger mitochondrial membrane depolarization, or increase apoptotic cell death, regardless of UVA exposure. Taken together, these results suggest that nano-TiO2 and UVA synergistically promote rapid ROS generation and MMP collapse, triggering apoptosis. Additionally, they show that small TiO2 particles are more phototoxic than larger ones.

Effects of Calcium Gluconate on Experimental Periodontitis and Alveolar Bone Loss in Rats

We examined the effects of calcium gluconate, an anti-inflammatory calcium salt, on ligature-induced experimental periodontitis and related alveolar bone loss. Calcium gluconate was orally administered daily for 10 days at 250, 125 or 62.5 mg/kg, beginning 1 day after ligation. We recorded changes in body-weight and alveolar bone loss and quantified the anti-inflammatory effects of calcium gluconate by measuring levels of myeloperoxidase (MPO), IL-1β and TNF-α. We also evaluated inducible nitric oxide synthase (iNOS) activity and malondialdehyde (MDA) concentration as a measure of antioxidant effects. Ligature placement produced a marked decrease in body-weight, increased alveolar bone loss, and led to increased MPO, IL-1β, TNF-α and MDA concentrations, as well as elevated iNOS activity, increased inflammatory cell infiltration and decreased collagen fibre content in gingival tissue. Histopathology revealed decreased alveolar bone volume, increased osteoclast cell numbers and activity, and an elevated percentage of osteclasts on the alveolar bone surface. The effects of ligature placement were significantly and dose-dependently inhibited by 10 days of daily oral treatment with 250 and 125 mg/kg of calcium gluconate. The results suggest that 10 days daily oral treatment with calcium gluconate effectively inhibits ligature placement-induced periodontitis and related alveolar bone loss via antioxidant effects.

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.

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.

A KOREAN FAMILY WITH A DOMINANTLY INHERITED β-THALASSEMIA DUE TO Hb DURHAM-N.C./BRESCIA [β114(G16)Leu→Pro]

We describe the molecular and the hematological characteristics of a Korean family with a dominantly inherited β-thalassemia. Carriers were characterized by moderate anemia, hypochromia, microcytosis, elevated Hb A2 and Hb F levels, and splenomegaly. DNA analysis revealed a CTG (Leu) to CCG (Pro) substitution at codon 114 of the β-globin gene, that leads to a highly unstable hemoglobin variant, Hb Durham-N.C./Brescia, and this was linked to the β haplotype V, [+−−−−+−], and framework 2. RNA analysis showed that the proband had comparable levels of mutant and normal β-mRNA. Translation of the mutant mRNA would give rise to non-functional hyperunstable β-globin chains, and their degradation would, by placing an additional burden on the proteolytic process of the red blood cell precursors, result in a more severe phenotype.