阿莫西林对人HepG2细胞DNA有短暂损伤作用

目的 为了探讨阿莫西林对人HepG2细胞DNA是否有损伤作用.方法 培养的人HepG2细胞经不同浓度(2、10、50和250μmol/L)阿莫西林处理1h或经50μmol/L阿莫西林处理不同时间(20、40、60、120和180min)后,运用单细胞凝胶电泳(SCGE)技术结合彗星图像软件(CASP)分析细胞尾部DNA百分率(tail DNA%)变化情况.结果 不同浓度阿莫西林处理后结果显示,HepG2细胞尾部DNA百分率明显升高,至50μmol/L阿莫西林时达到最高值,各浓度处理组与不处理对照组相比差异皆有显著性(P<0.01);而同一浓度(50μmol/L)阿莫西林处理不同时间后结果显示,HepG2细胞尾部DNA百分率逐渐升高,至1h处理时间点时达到最高值,其后随着处理时间延长HepG2细胞尾部DNA百分率逐渐降低.结论 阿莫西林对人HepG2细胞DNA有短暂损伤作用,阿莫西林诱发的HepG2细胞DNA损伤可能随时间延长逐渐被HepG2细胞本身修复除去.     

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.     

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells

Abstract

We investigated the genotoxic responses to two types of TiO₂ nanoparticles (<25 nm anatase: TiO₂-An, and <100 nm rutile: TiO₂-Ru) in human hepatoma HepG2 cells. Under the applied exposure conditions the particles were agglomerated or aggregated with the size of agglomerates and aggregates in the micrometer range, and were not cytotoxic. TiO₂-An, but not TiO₂-Ru, caused a persistent increase in DNA strand breaks (comet assay) and oxidized purines (Fpg-comet). TiO₂-An was a stronger inducer of intracellular reactive oxygen species (ROS) than TiO₂-Ru. Both types of TiO₂ nanoparticles transiently upregulated mRNA expression of p53 and its downstream regulated DNA damage responsive genes (mdm2, gadd45α, p21), providing additional evidence that TiO₂ nanoparticles are genotoxic. The observed differences in responses of HepG2 cells to exposure to anatase and rutile TiO₂ nanoparticles support the evidence that the toxic potential of TiO₂ nanoparticles varies not only with particle size but also with crystalline structure.     

Malathion‐induced oxidative stress,cytotoxicity, and genotoxicity in human liver carcinoma (HepG2) cells

Malathion is an organophosphate pesticide that is known for its high toxicity to insects and low to moderate potency to humans and other mammals. Its toxicity has been associated with the inhibition of acetylcholinesterase activity, leading to the interference with the transmission of nerve impulse, accumulation of acetylcholine at synaptic junctions, and subsequent induction of adverse health effects including headache, dizziness, nausea, vomiting, bradycardia, and miosis. Oxidative stress (OS) has been reported as a possible mechanism of malathion toxicity in humans. Hence, the aim of this study was to examine the role of OS in malathion‐induced cytotoxicity and genotoxicity. To achieve this goal, MTT, lipid peroxidation, and single cell gel electrophoresis (Comet) assays were performed, respectively, to evaluate the levels of cell viability, malondialdehyde (MDA) production, and DNA damage in human liver carcinoma (HepG₂) cells. Study results indicated that malathion is mitogenic at lower levels of exposure, and cytotoxic at higher levels of exposure. Upon 48 h of exposure, the average percentages of cell viability were 100% ± 11%, 117% ± 15%, 86% ± 15%, 35% ± 9%, and 27% ± 7% for 0, 6, 12, 18, and 24 mM, respectively. In the lipid peroxidation assay, the concentrations of MDA produced were 12.55 ± 0.16, 20.65 ± 0.27, 31.1 ± 0.40, 34.75 ± 0.45, and 15.1 ± 0.20 μM in 0, 6, 12, 18, and 24 mM malathion, respectively. The Comet assay showed a significant increase in DNA damage at the 24 mM malathion exposure. Taken together, our results indicate that malathion exposure at higher concentrations induces cytotoxic and genotoxic effects in HepG₂ cells, and its toxicity may be mediated through OS as evidenced by a significant production of MDA, an end product of lipid peroxidation. © 2009 Wiley Periodicals, Inc. Environ Toxicol 2010.     

Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells.

Curcumin is extensively used as a spice and pigment and has anticarcinogenic effects that could be linked to its antioxidant properties. However, some studies suggest that this natural compound possesses both pro- and antioxidative effects. In this study, we found that curcumin induced DNA damage to both the mitochondrial and nuclear genomes in human hepatoma G2 cells. Using quantitative polymerase chain reaction and immunocytochemistry staining of 8-hydroxydeoxyguanosine, we demonstrated that curcumin induced dose-dependent damage in both the mitochondrial and nuclear genomes and that the mitochondrial damage was more extensive. Nuclear DNA fragments were also evident in comet assays. The mechanism underlies the elevated level of reactive oxygen species and lipid peroxidation generated by curcumin. The lack of DNA damage at low doses suggested that low levels of curcumin does not induce DNA damage and may play an antioxidant role in carcinogenesis. But at high doses, we found that curcumin imposed oxidative stress and damaged DNA. These data reinforce the hypothesis that curcumin plays a conflicting dual role in carcinogenesis. Also, the extensive mitochondrial DNA damage might be an initial event triggering curcumin-induced cell death.     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

The role of oxidative stress in deoxynivalenol-induced DNA damage in HepG2 cells

Deoxynivalenol (DON) is a trichothecene mycotoxin and a cereals contamination, whose cytotoxicity has been shown in animals and various cells. However, with respect to the deoxynivalenol-induced DNA damage, especially in humans, are not well understood. The aim of this study was to assess the role of oxidative stress in deoxynivalenol-induced DNA damage, using human hepatoma HepG2 cells. Exposure of the cells to DON caused significant increase of DNA migration in comet assay at concentrations of 3.75-30 μM, which suggests that DON caused DNA strand breaks. To elucidate the role of antioxidation in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in comet assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. The DNA damage induced by DON was almost completely prevented. In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. Significant increase in the level of ROS was observed in HepG2 cells at a higher concentration (60 μM). The involvement of lipid peroxidation in the DNA damage of DON was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), the doses being 7.5-60 μM and 3.75-15 μM, respectively. These results indicate that the DNA damage induced by DON in HepG2 cells is probably related to the oxidative stress.     

Comparative evaluation in vitro of the herbicide flurochloridone by cytokinesis‐block micronucleus cytome and comet assays

The in‐vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO‐K1) cells. The cytokinesis‐block micronucleus cytome (CBMN‐cyt) and single‐cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25–15 μg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 μg/mL. Furthermore, 10 and 15 μg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 μg/mL of both flurochloridone and Rainbow® but only when 15 μg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single‐strand DNA breaks in vitro on mammalian cells. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 884–892, 2014.     

亚硝胺化合物对人源肝癌细胞HepG2的DNA损伤风险评价

目的 使用人肝癌HepG2细胞筛选亚硝胺的体外彗星试验的S9 mix配方，并对17种常见的亚硝胺化合物开展彗星试验，研究其DNA亲和力和碱基嵌入风险。方法 在非S9活化和S9活化条件下对HepG2细胞进行N-二甲基亚硝胺（NDMA）、N-二乙基亚硝胺（NDEA）、N-亚硝基二丁胺（NDBA）、N-亚硝基二异丙胺（NDIPA）、N-亚硝基-N-甲基-4-氨基丁酸（NMBA）、N-亚硝基甲乙胺（NMEA）、N-亚硝基-N-乙基异丙胺（NEIPA）、N-亚硝基二丙胺（NDPA）、N-甲基-N-亚硝基苯胺（NMPA）、亚硝基二苯胺（NDPh）、二乙醇亚硝胺（NDELA）、亚硝基吗啉（NMOR）、亚硝基-N-甲基-N-（2-苯基）乙胺（NMPEA）、亚硝基吡咯烷（NPYR）、亚硝基哌啶（NPIP）、4-甲基亚硝胺基-1-3-吡啶基-1-丁酮（NNK）、N-亚硝基降烟碱（NNN）给药处理，2种条件均设置溶媒对照（0.5% DMSO）、3个浓度梯度的给药组和阳性对照组，在非S9活化条件下以甲基磺酸甲酯（MMS）为阳性对照，S9活化条件下以环磷酰胺（CP）为阳性对照。以NDMA和NDEA为例比较3种S9 mix配方对亚硝胺化合物体外DNA亲和力和DNA损伤风险，选择效果最优者开展剩余化合物在S9条件下的彗星试验，计算各组尾DNA含量百分率（% tail DNA）的平均值和中位数。结果 在非S9代谢活化条件下17种常见亚硝胺化合物均未导致HepG2细胞核DNA明显损伤。S9 mix配方C中S9体积分数仅为3.36%，但对亚硝胺化合物的代谢活化效果最佳。在该条件下，除NDPh外，其余亚硝胺化合物均对HepG2细胞存在DNA的损伤作用。烷基类亚硝胺化合物对DNA损伤作用强弱顺序依次为NDMA>NEIPA>NDPA>NMEA>NDEA>NDBA>NDIPA，与化合物α氢的数目基本呈正相关。含苯基的亚硝胺化合物对DNA损伤作用强弱顺序依次为NMPEA>NMPA>NDPh，而环状亚硝胺化合物对DNA损伤作用强弱顺序为NMOR>NPIP≈NPYR。结论 提供最新的亚硝胺化合物体外DNA损伤风险数据，并提出适宜亚硝胺化合物的体外彗星试验S9 mix配方，为亚硝胺化合物的毒性评价提供手段。     

Relationship between reactive oxygen species and sodium-selenite-induced DNA damage in HepG2 cells

Selenium compounds, as an effective chemopreventive agent, can induce apoptosis in tumor cells. Reactive oxygen species (ROS) are important mediators in apoptosis induced by various stimuli, which include chemopreventive agents. In this study, we investigated the relationship between ROS and the levels of DNA damage induced by selenite in HepG2 cells. After HepG2 cells were treated with selenite, there was a dose-dependent decrease in cell viability. The levels of ROS induced by selenite were measured by 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) fluorescence, which shos a dose-and time-dependent increase in HepG2 cells. The levels of DNA damage in HepG2 increased in all cells treated with an increasing dose of selenite at 0, 2.5, 5, 10, and 20 μmol/L. N-acetylcysteine (NAC), a known antioxidant, increased cell viability and decreased ROS generation. Moreover, NAC effectively blocked DNA damage induced by selenite. These results revealed that ROS might play an important role in selenite-induced DNA damage that can be reduced by NAC treatment.     

Involvement of enniatins-induced cytotoxicity in human HepG2 cells

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A.     

Furazolidone induced oxidative DNA damage via up-regulating ROS that caused cell cycle arrest in human hepatoma G2 cells

Furazolidone (FZD) is an antimicrobial agent that has been shown to have mutagenic, genotoxic and potentially carcinogenic properties when tested in a variety of systems in vitro and in vivo. In this study, we investigated FZD's DNA damaging effect in human hepatoma cells aiming at further defining the molecular mechanism of FZD's cytotoxicity. Addition of FZD resulted in cell growth suppression and cell cycle arrest in S phase accompanied by remarkable DNA strand breaks with increased levels of intracellular reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine. Activities of antioxidases were down-regulated following FZD treatment and antioxidant agent catalase and superoxide dismutase ameliorated FZD's DNA damaging effects. Moreover, FZD caused much more extensive damage to mitochondrial DNA (mtDNA) than to nuclear DNA for which the decrease in mtDNA content correlated with FZD usage in a dose-dependent manner. However, there was no evidence of FZD induced mtDNA mutation in the mitochondrial DNA displacement loop. These results demonstrate that FZD up-regulates the production of intracellular ROS to cause oxidative DNA damage with mtDNA being the most vulnerable targets. Oxidative stress and the injury of mtDNA could be early indicators of FZD-induced cytotoxicity, with the resulting abnormal progression of the cell cycle.     

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by fluorescent microscopy of cytochalasin B‐blocked micronucleus formation

As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20‐nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20‐nm nanosilver solution determined by the inductively coupled plasma–mass spectrometry (ICP‐MS) analysis was 0.962 mg ml^?1. Analysis by ICP‐MS and TEM demonstrated the uptake of 20‐nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B‐blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration‐ and time‐dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml^?1 in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

The role of oxidative stress in Sudan IV‐induced DNA damage in human liver‐derived HepG2 cells

Objectives: We evaluated the role of oxidative stress in Sudan IV‐induced DNA damage, using human liver‐derived HepG2 cells. Methods: The DNA damaging effects of Sudan IV in HepG2 cells were evaluated by alkaline single cell gel electrophoresis assay and micronucleus test (MNT). To clarify the underlying mechanisms, we monitored the intracellular generation of reactive oxygen species (ROS) by 2, 7‐dichlorofluorescein diacetate assay and the level of oxidative DNA damage by immunoperoxidase staining for 8‐hydroxydeoxyguanosine (8‐OHdG). Furthermore, the intracellular glutathione (GSH) level was moderated by pretreatment with buthionine‐(S,R)‐sulfoximine (BSO), a specific GSH synthesis inhibitor. Results: A significant dose‐dependent increment in DNA migration was detected at all tested concentrations (25–100 μM) of Sudan IV. And in the MNT, a significant increase of the frequency of micronuclei was found at higher tested concentrations (50–100 μM). The data suggested that Sudan IV caused DNA strand breaks and chromosome breaks. In addition, significantly increased levels of ROS, 8‐OHdG formation were observed in HepG2 cells. It was also found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to Sudan IV‐induced DNA damage. Conclusions: Based on these data we believe that Sudan IV exerts toxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

吡非尼酮对肝癌HepG2细胞增殖和凋亡的影响(“豪森杯”优秀论文三等奖)

目的：研究吡非尼酮（pirfenidone,PF）对人肝癌细胞系HepG2增殖和凋亡的影响。方法：CCK-8法测定不同浓度PF对HepG2细胞增殖活性的影响;Hoechst 33258荧光染色法观察PF处理后HepG2细胞形态的变化;流式细胞仪检测细胞凋亡率。结果：PF对HepG2细胞具有显著增殖抑制作用,并呈浓度和时间依赖性;Hoechst 33258染色可见PF处理后细胞出现典型的凋亡形态学变化;流式细胞仪检测结果显示,与空白组比较,PF处理后的HepG2细胞凋亡率显著增加（P﹤0.01）。结论：PF对人肝癌细胞系HepG2细胞增殖具有抑制作用,且与诱导HepG2细胞凋亡有关。     

Organic extracts of coke oven emissions can induce genetic damage in metabolically competent HepG2 cells

Coke oven emissions (COEs) containing various carcinogenic polycyclic aromatic hydrocarbons (PAHs) represent the coal-burning pollution in the air. Organic pollutants in the aerosol and particulate matter of COEs were collected from the bottom, side, and top of a coke oven. The Comet assay and cytokinesis-block micronucleus cytome assay were conducted to analyze the genetic damage of extractable organic matter (EOM) of COEs on HepG2 cells. All the three EOMs could induce significant dose-dependent increases in Olive tail moment, tail DNA, and tail length, micronuclei, nucleoplasmic bridges, and nuclear buds frequencies, which were mostly positively correlated with the total PAHs concentration in each EOM. In conclusion, EOMs of COEs in the three typical working places of coke oven can induce DNA strand breaks and genomic instability in the metabolically competent HepG2 cells. The PAHs in EOMs may be important causative agents for the genotoxic effects of COEs.     

Zinc inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in human hepatocytes (HepG2 cells)

Aflatoxin B1 (AFB1) has strong carcinogenicity. Consumption of AFB1-contaminated agricultural products and the occurrence of hepatocellular carcinoma have received widespread attention. The aim of this paper was to investigate whether zinc supplementation could inhibit AFB1-induced cytotoxicity and genotoxicity in HepG2 cells and the mechanism of this inhibition. Our data suggest that zinc sources can relieve a certain degree of AFB1-induced cytotoxicity and genotoxicity by protecting against apoptotic body formation and DNA strand breaks, affecting S phase cell cycle arrest, reducing 8-OHdG formation, inhibiting global DNA hypomethylation and regulating gene expression in antioxidation, zinc-association and apoptosis processes. Consequently, zinc stabilizes the integrity of DNA and improves cell survival.These data provides new insights into the protective role of zinc in alleviating AFB1-induced cytotoxicity and mediating epigenetic changes in hepatocytes, demonstrating that zinc sources have detoxification properties in mycotoxin-induced toxicity.     

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.     

Influence of aroclor 1254 on benzo(a)pyrene-induced DNA breakage, oxidative DNA damage, and cytochrome P4501A activity in human hepatoma cell line

Both polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. They coexist widely in the environment at very low levels. Numerous studies indicated that aroclor1254 (one of PCBs mixture) is the inducer of cytochrome P450 1A enzyme acitivity. Benzo(a)pyrene (BaP) can cause a variety of toxicities in vitro, such as oxidative DNA damage and genotoxicity. In the present study, HepG2 cells were treated with either BaP (50 microM) or aroclor1254 at concentrations of 11.5 (low), 23.0 (medium), and 46.0 microM (high) alone, or pretreated the cells with aroclor1254 (11.5, 23.0, and 46.0 microM), followed by BaP (50 microM). It was found that 7-ethoxyresorufin-O-deetylase (EROD) activities of HepG2 cells exposed to either BaP or aroclor 1254 increased. DNA damage measured by DNA migration and the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) also increased in cells exposed to BaP, but not in cells exposed to aroclor1254. Under the Aroclor 1254 pretreatment condition, BaP-induced EROD activities was enhanced in cells exposed to the medium and high concentrations of aroclor1254 (P < 0.01 for both), whereas in all pretreatment groups aroclor1254 significantly increased BaP-induced DNA migration (P < 0.01 for all) and the 8-OHdG formation (P < 0.05 for all). In addition, there was positive correlation between the EROD induction activity and Olive tail moment (r(2) = 0.958, P < 0.01) or the levels of 8-OHdG (r(2) = 0.992, P < 0.01). The findings suggest that under the experimental conditions aroclor1254 may enhance BaP-induced DNA migration and oxidative DNA damage in HepG2, due to inducing CYP1A enzyme activity.     

苦荞内生真菌产物EE-2抑制HepG2生长及诱导其细胞凋亡

目的研究苦荞内生真菌KQH-2代谢醇提物EE-2对人肝癌细胞的抑制作用及其作用机制。方法采用MTT法考察EE-2对人肝癌细胞HepG2体外增殖的抑制作用,显微镜观察EE-2对HepG2细胞形态的影响,琼脂糖凝胶电泳检测细胞DNA完整性,并利用流式细胞仪检测HepG2细胞凋亡及细胞周期变化情况。结果用EE-2处理过的HepG2细胞,显微镜观察可见细胞脱壁圆缩,出现凋亡小体;细胞核降解。流式细胞仪检测发现,处理组的DNA直方图上有比对照组加强的SubG1峰,且可将HepG2细胞阻滞于G0/G1期。结论苦荞内生真菌KQH-2代谢醇提物EE-2可诱导人肝癌HepG2细胞的凋亡,且具有细胞周期阻滞作用。