Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin     

Water content in cultured mammalian cells for dosimetry of beta-rays from tritiated water

One of the critical factors for dosimetry of beta-rays from tritiated water is the water content within the cell. We estimated the cellular water concentration in cultured mammalian cells by measuring accurately the fraction of the extracellular water in the cell sample with [14C]inulin. The net water content (ml.g-1) after correcting for the extra-cellular water fraction was 0.858 for HeLa (human), 0.833 for JTC12P3 (monkey), 0.829 for NRK (rat), 0.843 for C3H10T1/2 (mouse) and 0.846 for L5178Y (mouse) cells. The mean water content (+/- S.E.M.) in these 5 lines of cultured mammalian cells was 0.842 +/- 0.005.     

Synthesis and antiproliferative activity of 3-aryl-2-(1H-benzotriazol-1-yl)acrylonitriles. Part III

A new series of 30 3-aryl-2-(1H-benzotriazol-1-yl)acrylonitriles were synthesized and tested for biological activity as part of our research in the antimicrobial and antitumor fields. In particular, title compounds were evaluated in vitro against representative strains of Gram-positive and Gram-negative bacteria (S. aureus, Salmonella spp), mycobacteria (M. fortuitum, M. smegmatis ATCC 19420 and M. tuberculosis ATCC 27294), yeast and mould (C. albicans ATCC 10231 and A. fumigatus). Furthermore, their antiretroviral activity against HIV-1 was determined in MT-4 cells together with cytotoxicity. In these assays title compounds and 47 additional derivatives described previously (P. Sanna, A. Carta, M.E. Rahbar Nikookar, Eur. J. Med. Chem. 35 (2000) 535-543; P. Sanna, A. Carta, L. Gherardini, M.E. Rahbar Nikookar, Farmaco 57 (2002) 79-87) were tested for their capability to prevent MT-4 cell growth. All compounds resulted devoid of antibacterial, antifungal and anti-HIV-1 activity. In anti-mycobacterial assays several compounds resulted active (MIC(50)=6.0-70 microM) against M. tuberculosis. However, since they showed cytotoxicity against MT-4 cells at lower concentrations (CC(50)=0.05-25 microM), their anti-mycobacterial activity was not selective. For this reason, the most cytotoxic compounds were also evaluated for antiproliferative activity against a panel of human cell lines derived from both hematological and solid tumors. Compound 34 resulted the most potent compound against the above human tumor-derived cell lines.     

Persisting learning deficits in rats after exposure to Pfiesteria piscicida.

Pfiesteria piscicida and other toxic Pfiesteria-like dinoflagellates have been implicated as a cause of fish kills in North Carolina estuaries and elsewhere. Accidental laboratory exposure of humans to P. piscicida has been reported to cause a complex syndrome including cognitive impairment. The current project was conducted to experimentally assess the possibility of cognitive effects of P. piscicida exposure in rats. Samples of water from aquaria in which P. piscicida zoospores were killing fish were frozen, a procedure that has been found to induce encystment. Thawed samples were injected into albino Sprague-Dawley rats. A significant learning impairment was documented in rats administered samples of P. piscicida that were recently frozen. Prolonged storage of Pfiesteria samples diminished the effect. No effect was seen in the recall of a previously learned task, but when the rats were called upon to learn a new task, the Pfiesteria-treated animals showed a significant learning deficit. This effect persisted up to at least 10 weeks after a single injection of Pfiesteria. The Pfiesteria-induced learning deficit did not seem to be associated with any obvious debilitation or health impairment of the exposed rats. Deficits in habituation of arousal and rearing behavior were detected using a functional observational battery. No Pfiesteria-induced effects on blood count and white cell differential or in a standard pathological screening of brain, liver, lung, kidney, and spleen tissue were seen at 2 months after exposure. These studies document a persistent learning impairment in rats after exposure to the dinoflagellate P.piscicida in otherwise physically well-appearing rats. This effect may partially model the symptoms of cognitive impairments that humans have shown after Pfiesteria exposure.     

Characterization of thymidine kinase in L5178Y mouse lymphoma cells

Thymidine kinase (TK) was isolated from four different cell lines (TK +/+ P4, TK +/- P4.3.4, TK +/- 3.7.2C, and TK -/- P4.3) that represent the genotypes of L5178Y mouse lymphoma cells. TK isolated from each of the different cell lines was characterized with respect to the estimated isoelectric point (pI), temperature sensitivity, estimated substrate dissociation constants (Km), estimated inhibitor constant (Ki), and response to the activator deoxycytidine diphosphate. The characteristics of TK from the different cell lines were compared to determine whether the product of the TK gene was changed by the mutation that produced the TK -/- genotype and reverse mutation to the TK +/- genotype. The results indicate that the TK enzymes isolated from the TK +/+ P4, TK +/- P4.3.4, and TK +/- 3.7.2C cells have similar, if not the same, characteristics. The small amounts of TK associated with TK -/- P4.3 and the mitochondria from TK +/+ P4 had similar characteristics, and both were different in many respects from the TK associated with the TK +/+ P4, TK +/- P4.3.4, and TK +/- 3.7.2C. These results raise a question about whether a structural gene is the target for chemical mutagens in the L5178Y TK +/- assay; however, this can only be answered by the isolation of the TK gene from each of the L5178Y genotypes and determination of the nucleotide sequence.     

Cytotoxic Activity of Piper cubeba Extract in Breast Cancer Cell Lines

This study aimed to evaluate the cytotoxicity of a crude extract of Piper cubeba against normal and breast cancer cell lines. To prepare the extract, P. cubeba seeds were ground, soaked in methanol and dichloromethane and isolated by column chromatography. Fractions were tested for cytotoxicity effects on normal fibroblast (L929), normal breast (MCF-12A) and breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The most effective fraction was selected for DNA fragmentation assay to detect apoptotic activity. The results showed that the methanolic crude extract had a higher cytotoxic activity against MDA-MB-468 and MCF-7 than a dichloromethane crude extract. Then, the methanolic crude extract was separated into six fractions, designated A to F. Fraction C was highly active against breast cancer cell lines with an IC₅₀ value less than 4 μg/mL. Therefore, Fraction C was further separated into seven fractions, CA to CG. The ¹H-NMR profile showed that Fraction CE was long chain hydrocarbons. Moreover, Fraction CE demonstrated the highest activity against MCF-7 cells with an IC₅₀ value of 2.69 ± 0.09 μg/mL and lower cytotoxicity against normal fibroblast L929 cells with an IC₅₀ value of 4.17 ± 0.77 μg/mL. Finally, DNA fragmentation with a ladder pattern characteristic of apoptosis was observed in MCF-7, MDA-MB-468, MDA-MB-231 and L929 cells, but not in MCF-12A cells.     

In vitro toxicity of welding fumes and their constituents

Welding fumes from a wide variety of processes and applications were assayed for toxicity with BHK21 cell line and SHE primary cells in culture. The most toxic fumes are those from the manual metal arc welding of stainless steel (MMA/SS) (LD50 = 7-14 microgram/ml), although all other welding fumes tested are toxic, with potencies lower by a factor of 10-200. The activity of MMA/SS is presumably due to the presence of high concentrations of Cr(VI) in the soluble fraction: For all other fumes the lowered activity (LD50 = 80-800 microgram/ml) is limited mostly to the insoluble fraction, and in part can be related to the presence of MnO2 and Fe3O4 which are toxic at such levels in these cell culture assays. Slight discrepancies between survival tests for the two cell lines, and between survival and lactate dehydrogenate release for BHK, indicate a differential response to certain constituents of these complex materials. These results suggest the need for a battery of different types of assays for use in an eventual ranking of exposures for the purpose of relative risk assessment.     

Citrus limonoids induce apoptosis in human neuroblastoma cells and have radical scavenging activity

Citrus limonoid glucosides, a family of fruit bioactive compounds, were postulated to have free radical-scavenging and apoptosis-inducing properties against certain types of cancers. Four highly purified limonoid glucosides, limoin 17beta D-glucopypranoside (LG), obacunone 17beta D-glucopyranoside (OG), nomilinic acid 17beta D-glucopyranoside (NAG), and deacetylnomilinic acid 17beta D-glucopyranoside (DNAG) were tested for superoxide radical (O(2)(-))-quenching activity and cytotoxic action against undifferentiated human SH-SY5Y neuroblastoma cells in culture. All 4 scavenged O(2)(-) as measured by inhibition of pyrogallol decomposition in a spectrophotometric assay. Quenching by NAG in particular emulated an equivalent concentration of vitamin C. When added to the medium of SH-SY5Y cells in culture, micromolar amounts of LG and OG, compared with untreated controls, caused a cessation of cell growth and rapid cell death (P < 0.001); NAG and DNAG were better tolerated, but nonetheless toxic as well. Cytotoxicity was related to a concentration- and time-dependent increase in caspase 3/7 activity, suggesting that limonoid glucosides were capable of inducing apoptosis. Arrested cell growth and the induction of apoptosis were confirmed by flow cytometry and DNA fragmentation analysis. Importantly, caspase induction at 12 h correlated with cell survival at 24 h (P = 0.046), suggesting that apoptosis was the primary cause of cell death. We conclude that citrus limonoid glucosides are toxic to SH-SY5Y cancer cells. Cytotoxicity is exerted through apoptosis by an as yet unknown mechanism of induction. Individual limonoid glucosides differ in efficacy as anticancer agents, and this difference may reside in structural variations in the A ring of the limonoid molecule.     

不同代谢活化系统增强苯并(a)芘诱导人支气管上皮细胞转化效应

目的探讨不同的代谢活化系统在苯并(a)芘[B(a)P]诱导人细胞转化模型中的应用。方法选择人支气管上皮细胞HBETR,采用3种不同的代谢活化方式:分别为加入大鼠S9组分(S9-Mix)、高表达代谢关键酶P450CYP1A1(HBETR-1A1细胞)和染毒前48小时用低剂量B(a)P诱导(HBETR-IN细胞)。通过软琼脂克隆形成试验和裸鼠皮下成瘤试验来比较在不同的代谢活化系统下,间接致癌物B(a)P诱导细胞转化的效能。结果通过蛋白印迹和酶活性检测结果验证HBETR-1A1细胞构建成功。细胞的生物学特性没有明显的改变。20μmol/LB(a)P染毒作用下HBETR-1A1、HBETR-IN细胞出现转化时间均为11周,而未加入活化系统时,HBETR细胞需14周才能获得恶性转化。HBETR细胞在加入和不加入S9-Mix的条件下,转化时间分别是14周、20周。上述转化的效果与CYP1A1酶活性以及蛋白表达水平相一致。结论三种代谢系统都能够促进间接致癌物B(a)P的代谢活化,缩短细胞的转化间期,提高细胞转化效率。从试验操作难度、试验的重复性及结果的可靠性等几个方面比较三种代谢系统应用的可行性,低剂量诱导作为新的代谢活化的方法在细胞转化试验中有着潜在的应用前景。     

Cytotoxic 2,6-bis(arylidene)cyclohexanones and related compounds

A number of 2-arylidenecyclohexanones 1, 2, 6-bis(arylidene)cyclohexanones 2 and related Mannich bases 3-5 were prepared. Various torsion angles as well as atomic charges on olefinic carbon atoms were determined by molecular modelling on all compounds. These molecules showed cytotoxicity towards murine P388 and L1210 cells as well as to human Molt 4/C8 and CEM T-lymphocytes. The average cytotoxicity of the dienones 2 was more than three times greater than was found with the monoarylidene analogues 1, and, in general, were slightly more cytotoxic than the Mannich bases 3-5. A number of the compounds displayed potency towards a panel of human tumour cell lines and most of the representative compounds in series 2-5 were selectively toxic to colon cancers and leukaemic cells.     

Self-assembly of novel molecular complexes of 1,10-phenanthroline and 5-amino-1,10-phenanthroline and evaluation of their in vitro antitumour activity

Novel molecular complexes of 1,10-phenanthroline (phen) and 5-amino-1,10-phenanthroline (5-NH2-phen) [(5-NH2-phen)2(phen) (H2O)3 (1), (phen)2(imidazole) (H+) (BF4-) (2), (phen)2(benzimidazole) (H+) (BF4-) (3), (5-NH2-phen)4(H2O)3 (4), and (phen)3 (indole) (H+) (BF4-) (5)] were synthesized via self-assembly processes and their in vitro anticancer activity was investigated. The structures of the compounds were confirmed by UV, FTIR, CIMS(CH4) and elemental analysis. The crystal structure of 2 was determined by X-ray diffraction. Cytotoxicity of the substances was measured using the cultivated human tumour cell lines HepG2, HEp-2, and 8-MB-GA. The tested substances showed different activity depending on the cell line and amount used. Substances 2 and 3 were not toxic to the non-tumour cells (Lep-3), but significantly toxic to all tumour ones. This is not the case with compounds 4 and 5, which are non-toxic towards carcinogenic cell lines, but even stimulate both HepG2 and HEp-2.     

Cytotoxic effects,alkylating properties and molecular modelling of coumarin derivatives and their phosphonic analogues

The cytotoxic effects and alkylating activity of a series of 3-[1-(alkylamino)-ethylidene]-chroman-2,4-dione (4a-4c), 2-methoxy-3-[1-(alkylamino)-ethylidene]-2,3-dihydro-2,4-dioxo-2lambda(5)-benzo[e][1,2] oxaphosphinane (5a-5c) and [2-oxo-4-phenyl(alkyl)-2H-chromen-3-yl]-phosphonic acids dimethyl ester (6a-6c) on the two leukemia cell lines HL-60 and NALM-6 have been determined. The test compounds are much more toxic to NALM-6 cells than to HL-60 cells. IC(50) data are up to nine times lower for the NALM-6 than for the HL-60 cell lines. As determined in an in vitro Preussmann test phosphonic derivatives 6a-6c possess very high (+++) alkylating activity, phosphoric derivatives 5a-5c are less active (++) while the derivatives 4a-4c can be included in the group of low activity (+) alkylating agents. Using regression analysis QSAR we found a relationship between biological activity and the physicochemical properties of the test compounds. Their cytotoxic effect increases with an increase of the hydrophobic parameters in the region of the substituents at the 2-, 3- and 4-positions of the benzopyrone skeleton of 4-6.     

Assessing the Detoxication Efficiencies of Wastewater Treatment Processes Using a Battery of Bioassays/Biomarkers

A battery of in vitro bioassays, including a Neutral Red (NR) assay using MCF-7 cells for predicting cytotoxic chemicals, an ethoxy resorufin-O-deethylase (EROD) activity assay using H4IIE cells to check for dioxin-like chemicals, and a recombinant gene yeast assay for screening estrogenic chemicals, was conducted to assess the removal efficiencies of trace toxic chemicals by different treatment processes in the waste water treatment plant (WWTP). The effluents were extracted by solid phase extraction (SPE) and were fractionated into three fractions based on polarities. The battery of bioassays was performed for each fraction. In the battery, the toxicities of the effluents were described according to their modes of actions (MOA) or biomarkers and the properties of the toxic chemicals were categorized by their polarities and MOAs. The proposed procedure could be used as a tool to diagnose the toxic characteristics of the complicate mixture. The results showed that cytotoxic, dioxin-like and estrogenic chemicals could be detected in all samples. In the influent, cytotoxic and dioxin-like chemicals were mainly in polar fraction and estrogenic chemicals were in non-polar and moderate-polar fractions. The secondary treatment (active sludge) could remove a small amount of these toxicants. Among different types of advanced treatments, flocculation was good enough to remove most of the cytotoxic chemicals and a combination of flocculation, ozone oxidation, and post-biological treatment could eliminate most of the dioxin-like and estrogenic chemicals.     

二十碳五烯酸（EPA）对胃癌细胞增殖与凋亡的影响

目的观察二十碳五烯酸（EPA）对人胃癌细胞系增殖与凋亡的影响,并探讨其作用机制。方法以终浓度为10、20、40μg／ml的EPA作用于SGC-7901和MGC-803人胃癌细胞系24-72h。采用四甲基偶氮唑蓝法检测细胞增殖抑制率,采用流式细胞技术分析细胞周期分布与细胞凋亡,利用荧光探针rhodamine 123测定线粒体膜电位,酶联免疫吸附法测定线粒体和胞浆中细胞色素C水平,荧光光谱法测定凋亡效应酶半胱天冬蛋白酶-3（caspase-3）活性。结果经10-40μg／ml EPA处理的胃癌细胞增殖率显著降低,且呈现时间依赖性。与对照组比较,经40g／ml EPA作用72h后,SGC-7901和MGC-803细胞G0／G1期细胞的比例均增加（P=0.006、P=0.009）。经40g／ml EPA作用24h后,胃癌细胞线粒体膜电位显著低于对照组（P=0.001、P=0.047）;线粒体内细胞色素C的含量明显少于对照组（P=0.001、P=0.000）,而细胞浆中细胞色素C含量显著高于对照组（P=0．001、P=0．000）。在SGC-7901细胞中,caspase-3活性随着EPA（40g／mL）作用时间延长而升高。结论EPA通过诱导细胞周期阻滞和激活线粒体通路,抑制人胃癌细胞增殖,诱导细胞凋亡。     

苯并(a)芘通过活化蛋白-1-细胞周期蛋白D1/E2F-1信号通路引起细胞周期的改变

目的 探讨活化蛋白-1(activated protein-1,AP-1)在苯并(a)芘[benzo(a)pyrene,B(a)P]引起的人胚肺成纤维细胞(HELF)周期改变中的作用以及AP-1与细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖激酶4(CDK4)和E2F-1/4的上下游关系.方法 转染AP-1荧光报告质粒的HELF细胞(AP-H)无血清培养48 h后,加入2μmol/L B(a)P作用24 h,用荧光检测法检测AP-1的相对活性.用AP-1的化学抑制剂姜黄素(curcumin)抑制其活性,观察它与cyclin D1/CDK4和E2F-1/4的上下游关系.采用流式细胞仪检测细胞周期的变化,用Western blot检测细胞中cyclin D1、CDK4和E2F-1/4蛋白水平的改变.结果 2 μmol/LB(a)P作用24 h后,G1期细胞比例由(71±2)%减少为(48±3)%,差异有统计学意义(P＜0.05);AP-1的活性增加;cyclin D1/E2F-1的蛋白含量增加;CDK4/E2F-4的蛋白水平没有明显改变.抑制AP-1活性后,B(a)P诱导的细胞周期的改变被逆转,B(a)P诱导的cyclin D1/E2F-1蛋白含量的增加被抑制,CDK4/E2F-4蛋白含量没有明显改变.结论 B(a)P通过AP-1引起HELF周期的改变.AP-1是cyclin D1/E2F-1的上游信号分子,但对CDK4/E2F-4的表达不具有调节作用.     

Lack of evidence for contact sensitization by Pfiesteria extract

BACKGROUND: Members of the estuarine dinoflagellate genus Pfiesteria are reported to have been responsible for massive fish kills in the southeastern United States. Some reports suggest that exposure to waters having Pfiesteria blooms or occupation-related exposure might result in Pfiesteria-induced dermal irritation and inflammation. Although the toxin has not been isolated and purified, the original data suggested both hydrophilic and hydrophobic toxic components. Some investigators propose that dermonecrotic properties are associated with a hydrophobic fraction. OBJECTIVES: A bioactive C18-bound putative toxin (CPE) extracted from Pfiesteria-laden aquarium water during active fish-killing conditions was examined in the present study to evaluate its potential to produce inflammation and dermal sensitization and to determine whether the inflammation and dermatitis reported in early human exposure studies were allergic or irritant in nature. RESULTS: This fraction was cytotoxic to mouse Neuro-2A cells and primary human epidermal keratinocytes (NHEK) at a concentration of 1 mg/mL. Balb/C mice exposed to 50-200% CPE by skin painting exhibited a 6-10% increase in ear swelling relative to vehicle-treated mice in a primary irritancy assay. There was no increase in lymph node cell proliferation as measured using the local lymph node assay. Exposure to CPE in culture up-regulated interleukin-8 in NHEK, whereas granulocyte macrophage-colony-stimulating factor and tumor necrosis factor alpha were only minimally altered. CONCLUSIONS: This study suggests that CPE is cytotoxic to keratinocytes in culture at high concentrations and that it induces mild, localized irritation but not dermal sensitization.     

Effect of colonic luminal components on induction of apoptosis in human colonic cell lines

Apoptosis is central to cell number regulation in the colonic epithelium, and interest in its role in colon carcinogenesis has been growing rapidly. It thus becomes of interest to characterize luminal components, possibly of dietary origin, that may influence this process. We have investigated the sensitivity of two human colonic cell lines, the human adenocarcinoma cell line (HT-29) and the human fetal colonic mucosa cell line (FHC), to induction of apoptosis by sodium butyrate, bile acids, and human fecal water fractions. The apoptotic effect has been studied by 1) morphological changes in cells examined by fluorescence microscopy, 2) DNA fragmentation analysis by gel electrophoresis, 3) flow cytometry analysis of DNA strand breaks assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL), and 4) poly(ADP-ribose) polymerase cleavage by Western blot. Sodium butyrate and bile acids induced a time- and concentration-dependent apoptosis in both cell lines. Quantitation of this effect, by use of the TUNEL assay, indicated that deoxycholic acid was most effective in inducing this effect at lower concentrations and at shorter times. Apoptotic effects were also observed, in both cell lines, when the cells were exposed to intact human fecal waters (the fecal fraction in direct contact with the epithelium) and their lipid extracts, with the intact samples being more effective. Although all fecal waters examined induced apoptosis, quantitation of the effect by the TUNEL assay indicated that the ability to induce apoptosis differed markedly between samples. Induction of apoptosis by the fecal waters was not correlated to cytotoxicity but was negatively correlated to the pH of the samples. Interestingly, the cells derived from the fetal mucosa (FHC) were consistently less sensitive to apoptotic effects of the luminal components than the tumor-derived cells (HT-29). Thus human fecal water fractions induce apoptosis in colonic cells, and this effect is not due to lipid components alone.     

Chemopreventive properties of trans-resveratrol against the cytotoxicity of chloroacetanilide herbicides in vitro

The beneficial effect of trans-resveratrol (RESV) on health is well documented. Our aim was to study the putative preventive effect of RESV on the cytotoxicity of frequently used herbicides (alachlor, acetochlor). Estrogen receptor positive (ER+) MCF-7 human mammary carcinoma, HepG2 (ER+) human hepatocellular carcinoma and VERO estrogen receptor negative (ER-) non-transformed monkey fibroblast cell lines were treated with alachlor and acetochlor (2-500 microg/ml) as toxic agents, and RESV (10 microM) as preventive agent. The MTT dye reduction assay was performed to test cytotoxicity, and flow cytometry to test cell proliferation and apoptosis. RESV is not cytotoxic in the concentration range of 1-100 microM on neither cell lines examined after 24 h, but cytotoxic on Vero and MCF-7 cells at 100 microM after 48h, and on all three cell lines after 72 h. On both ER+ cell lines a stimulation of viability occurs in the low concentration range (0.5-12.5 microM) as detected by the MTT assay. Cell cycle analysis of the culture shows a significant increase of S-phase cells at low concentrations of RESV (10-50 microM) and a decrease in the 100-200 microM concentration range. The ratio of apoptotic cells significantly increases after the administration of 50 microM RESV, depending on the incubation time. The cytotoxicity of 20-65 microg/ml alachlor and 10-65 microg/ml acetochlor was significantly decreased by the addition of 10 microM RESV in Vero ER- cells whereas no significant change was detected on ER+ cell lines MCF-7 and HepG2. These results show that RESV protects non-transformed ER- cells, but has no such effect on ER+ tumor cells.     

Cyelin D1和CDK4正性调节苯并(a)芘所致人胚肺成纤维细胞周期改变

目的 研究苯并(a)芘[B(a)P]对人胚肺成纤维细胞(HELF)的细胞周期分布及细胞周期蛋白D1(cyclin D1)和细胞周期蛋白依赖激酶4(CDK4)蛋白表达的影响,并探讨两种蛋白含量改变与细胞周期效应之间的关系.方法 将反义cychn D1质粒和反义CDK4质粒导入HELF细胞内.建立两种质粒稳定转染的细胞模型.用0.1、0.5、2.5和12.5μmol/L的B(a)P处理HELF细胞24h.用蛋白印迹方法检测cvclin D1和CDK4蛋白表达水平;利用流式细胞技术检测B(a)P处理对HELF细胞及两种稳定转染细胞系细胞周期的影响.结果 成功建立了反义cyelin D1和反义CDK4稳定转染的细胞系.不同剂量B(a)P处理可引起cvclin D1蛋白表达的显著增加,但对CDK4蛋白表达无明显影响;2.5/μmol/L的B(a)P处理HELF细胞24h后,引起其细胞周期G1期显著下降,S期显著增加;2.5μmol/L的B(a)P处理反义cyclin D1和反义CDK4稳定转染的HELF细胞24h后,对其细胞周期的分布无显著影响.结论 Cyelin D1和CDK4基因均参与了B(a)P所致细胞周期改变过程,并发挥正性调节作用.