Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes.

The toxicity of hexavalent chromium and nickel was investigated using primary cultures of hepatocytes as an in vitro system. Cr VI and Ni are widely used in the steel and orthopaedic implant industry. Although their toxicity has been extensively investigated, the mechanism(s) of action is/are not fully understood. Monolayer cultures of hepatocytes (10(5) cells/cm2) were exposed to various concentrations of Cr VI and Ni for 24 h. Cells were stained with phalloidin-FITC for the detection of the cytoskeletal component, F-actin, and Annexin V-FITC and propidium iodide for the detection of the mode of cell death. Exposure of cells to Cr VI (1, 5, 10 and 50 microM) resulted in the loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Ni, on the other hand, induced detectable damage to the cytoskeleton only at 500 microM. Staining of the cells with Annexin V and propidium iodide showed that Cr VI induces apoptosis at low concentrations (5 microM), and necrosis at higher concentrations (25 and 50 microM). Ni almost exclusively induced necrosis at 500 microM with very few cells undergoing apoptosis. Below this concentration it had no discernable effect on hepatocytes. Damage to the cell cytoskeleton caused by Cr VI may be an early indication of apoptosis in hepatocytes.     

Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: A mechanism for its cytotoxic activity

Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC₅₀ ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules.     

The cathepsin B inhibitor,z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis.     

Selective inhibition of cytokinesis in sea urchin embryos by low concentrations of stypoldione, a marine natural product that reacts with sulfhydryl groups

Stypoldione is a marine natural product that inhibits cells division in marine embryos and in mammalian cell cultures. The mechanism responsible for the ability of the compound to inhibit cell division is not known. The compound was found in early studies to inhibit polymerization of tubulin into microtubules in vitro, which lead to the suggestion that inhibition of microtubule polymerization in cells might be responsible for the ability of the compound to inhibit cell division. More recently, stypoldione was found to react covalently with the sulfhydryl groups of a number of proteins including tubulin and with sulfhydryl groups of peptides and small molecules. Thus, stypoldione could potentially react with a large number of cellular targets. In the present study, we have examined the effects of stypoldione on the organization of microtubules and chromatin in cells, in relation to the ability of the compound to inhibit cell division. We used indirect immunofluorescence light microscopy of fixed and stained sea urchin embryos during the first and second divisions after fertilization, with stains specific for tubulin and DNA. We found that stypoldione exerted qualitatively different effects on cell division and microtubule organization and function at different concentrations. At the lowest effective concentrations, 5-10 microM, stypoldione selectively inhibited cytokinesis. Mitotic division occurred normally, usually with no discernible perturbation of microtubule organization or function, and cells became multinuclear. At somewhat higher concentrations, 20-40 microM, stypoldione blocked embryos before streak stage of the first division and, although microtubules were present, their organization was perturbed and they often formed unusual "spiral aster" arrays. At 80 microM and above, microtubules in blocked cells were largely absent. Thus, stypoldione uncouples cytokinesis from mitosis at the lowest effective concentrations and, although it can disrupt microtubules at relatively higher concentrations, it inhibits cell division at the lowest effective concentrations by a selective action on cytokinesis through a mechanism that does not appear to involve disassembly of microtubules.     

去甲斑蝥素对体外微管蛋白聚合的抑制作用

目的探讨去甲斑蝥素(NCTD)对微管蛋白聚合-解聚过程的影响。方法采用蛋白凝胶电泳鉴定从猪脑中提取的微管蛋白,并用Bradford法测定微管蛋白含量。在经过秋水仙碱和紫杉醇验证稳定的37℃和4℃平衡体系内加入NCTD 10,20和30μmol.L-1观察微管蛋白聚合-解聚活性。人卵巢癌SK-OV-3细胞加入NCTD 60μmol.L-1作用8 h,取上清和沉淀,Western印迹法检测微管蛋白含量。结果与空白对照组相比,在37℃聚合反应中,NCTD 10,20和30μmol.L-1组对微管蛋白聚合的抑制率分别为(5.5±2.7)%,(7.1±1.2)%和(27.5±0.4)%,在此浓度范围内NCTD随浓度的升高抑制作用增强(P<0.05),但对微管蛋白在4℃的解聚影响不显著。NCTD 60μmol.L-1作用人卵巢癌细胞后,细胞中游离微管蛋白增加了(91.5±8.5)%,聚合微管蛋白量减少了(51.8±3.8)%,说明NCTD抑制了人卵巢癌细胞SK-OV-3中微管蛋白的聚合。结论 NCTD对体外微管蛋白的聚合具有一定的抑制作用。     

Effects of benzimidazole analogs on cultures of differentiating rodent embryonic cells.

Micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were employed to assess the in vitro developmental toxicity of the benzimidazole analogs, mebendazole (MBZ), thiabendazole (TBZ), and nocodazole (NCZ), in addition to the classic microtubule inhibitor, colchicine. Comparison was made to albendazole (ABZ), a previously studied benzimidazole anthelmintic. Two parameters for assessing developmental toxicity were measured: differentiation and cytotoxicity. The relative potencies of the benzimidazole analogs in the micromass system (NCZ greater than MBZ approximately ABZ much greater than TBZ) mirrored their effectiveness in an assay for in vitro inhibition of mammalian tubulin polymerization. Colchicine also exhibits a high affinity for mammalian tubulin and was a potent inhibitor of cell proliferation, chondrogenesis, and neuronal differentiation. Immunofluorescent staining of Day 1 LB cultures with a monoclonal antibody to beta-tubulin revealed that these agents elicited mitotic arrest. Many anti-tubulin agents are teratogenic in rats and their in vivo developmental toxicity may reflect perturbation of microtubular structure or function. With the exception of TBZ, these agents should be considered potential developmental toxicants since they inhibit cell growth and differentiation of micromass cultures at nanomolar concentrations.     

秋水仙碱对大鼠肾细胞NRK体外毒性机制初步探讨

目的:考察秋水仙碱体外肾毒性及其主要机制。方法:体外培养大鼠肾细胞NRK,通过MTT试验、细胞形态学改变、乳酸脱氢酶释放率、Hoechst/PI双染色法、流式细胞术考察秋水仙碱对大鼠肾细胞NRK是否具有毒性作用,进而阐明秋水仙碱肾毒性产生的可能机制。结果:秋水仙碱在0.1~10μmol/L浓度范围内,对NRK细胞的抑制率呈浓度依赖性。1μmol/L处理组细胞形态学发生明显改变,乳酸脱氢酶释放率随着给药浓度的增加也逐渐增加。Hoechst/PI双染荧光观察10μmol/L处理组细胞处于晚期凋亡及坏死状态,Annexin V-PI双染流式检测细胞凋亡率随着给药浓度的增加而升高。结论:秋水仙碱在0.1~10μmol/L的浓度范围内具有较强的体外肾毒性,其机制可能是秋水仙碱将细胞阻滞在G2/M期,影响细胞的分裂从而诱导细胞凋亡,对细胞产生毒性。     

文蛤多肽抑制肿瘤细胞微管蛋白聚合

目的探讨文蛤多肽(Mere15)抑制人肺癌A549细胞增殖作用及其机制。方法 MTT比色法检测细胞增殖抑制率;流式细胞仪分析细胞周期分布的变化;微管蛋白免疫荧光检测技术检测Mere15对微管蛋白聚合的影响;Western blot检测Mere15对p21蛋白表达的影响。结果 Mere15可抑制A549细胞生长,抑制率呈剂量和时间依赖性;随着处理时间的增加,A549细胞G2/M期比例逐渐升高,微管蛋白聚合受到抑制,p21蛋白表达水平逐渐升高。结论 Mere15具有抑制A549细胞增殖的作用,其机制可能与抑制微管蛋白聚合有关。     

2,5-Hexanedione alters microtubule assembly. I. Testicular atrophy, not nervous system toxicity, correlates with enhanced tubulin polymerization

Charles River CD rats (200 g) were divided into three groups receiving either 1% 2,5-hexanedione (2,5-HD) or 0.035% 3,4-dimethyl-2,5-hexanedione (DMHD) in the drinking water or water alone (control) for 4 weeks. The two treated groups experienced similar nervous system dysfunction and systemic toxicity. Testicular toxicity, as evidenced by histological changes and decreased testis weight, was present only in 2,5-HD-treated rats. Tubulin was purified from brain and testis and assembly properties were determined. Purified brain and testis tubulin derived from the 2,5-HD-intoxicated rats displayed altered assembly with a shortened nucleation phase and more rapid rate of elongation. Brain tubulin from DMHD-intoxicated rats displayed assembly behavior similar to controls, while testis tubulin from DMHD-intoxicated rats displayed assembly behavior intermediate between the control and 2,5-HD tubulin preparations. The presence of gamma-diketone-induced assembly alterations following in vivo intoxication was accompanied by the formation of a high-molecular-weight protein identified as crosslinked tubulin. From these data, we conclude that microtubule assembly alterations are not etiologic in the development of nervous system dysfunction following intoxication but may represent the biochemical mechanism of 2,5-HD-induced testicular atrophy.     

Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse‐XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady‐state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady‐state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.     

Tris (1,3‐dichloro‐2‐propyl) phosphate induces toxicity by stimulating CaMK2 in PC12 cells

Tris (1,3‐dichloro‐2‐propyl) phosphate (TDCIPP) is one of the widely used organophosphorus flame retardants (OPFRs), which are regarded as suitable substitutes for brominated flame retardants (BFRs). Previously, we have validated the toxicity of TDCIPP in PC12 cells owing to the induced alterations in GAP43, NF‐H, CaMK2a/2b, and tubulin α/β proteins; however, limited information is currently available on the toxicity and mechanism of TDCIPP. In the present study, cytotoxicity effects were evaluated by exposing PC12 cells to different concentrations of TDCIPP (0–50 μM) for 4 days. To explore the possible mechanisms through which cytotoxicity is induced, changes in intracellular [Ca²⁺]_i levels and the activation of calmodulin dependent protein kinase 2 (CaMK2), c‐Jun N‐terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 mitogen‐activated protein kinases (MAPK) pathways were evaluated. Furthermore, PC12 cells were pretreated with CaMK2 inhibitor KN93 to investigate the relationship between TDCIPP‐induced phosphorylation of CaMK2 and activation of JNK, ERK1/2, and p38 MAPK pathways. Our results indicate that TDCIPP‐induced toxicity might be associated with the overload of [Ca²⁺]_i levels, increased phosphorylation of CaMK2, and activation of the JNK, ERK1/2, and p38 MAPK pathways, the lattermost of which was further demonstrated to be partially elicited by the CaMK2 phosphorylation.     

The role of GTP Binding and microtubule-associated proteins in the inhibition of microtubule assembly by carbendazim.

The fungicide carbendazim (CBZ) is known to disrupt microtubular structures in the testis and to cause testicular toxicity in rats. To investigate the mechanism underlying the toxicity of CBZ, tubulin and microtubule-associated proteins (MAPs) were isolated from rat testis and brain using two techniques. The effects of CBZ on MT assembly were compared with the known microtubule (MT) disruptors, colchicine and nocodazole. CBZ (100 microM) had no effect on the assembly of MTs from MAP-containing tubulin isolated with one cycle of glycerol-dependent assembly and disassembly while colchicine (40 microM) and nocodazole (12.5 microM) strongly inhibited the assembly reaction. Similarly, formation of MTs from tubulin prepared with two cycles of glycerol-dependent assembly was strongly inhibited by colchicine and nocodazole but only weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP-free tubulin isolated with glutamate. However, the inhibition by CBZ was reversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled MTs caused immediate depolymerization, whereas CBZ did not directly cause depolymerization. However CBZ was an effective inhibitor of the polymerization of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data suggest that CBZ interferes with initial events of MT polymerization, specifically GTP binding, and that MAPs moderate this effect.     

Reduced tubulin tyrosination as an early marker of mercury toxicity in differentiating N2a cells.

The aims of this work were to compare the effects of methyl mercury chloride and thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated alpha-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total alpha-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth.     

Microtubule-disrupting effects of gallium chloride in vitro.

Gallium chloride (GaCl3), an antitumor agent with antagonistic action on iron, magnesium and calcium, was tested for its ability to alter the polymerization of purified tubulin (2.2 mg/ml) in a cell-free system in vitro. GaCl3 (250 microM) does not mimic the effect of 10 microM paclitaxel and, therefore, is not a microtubule (MT)-stabilizing agent that can promote tubulin polymerization in the absence of glycerol and block MT disassembly. In contrast, GaCl3 mimics the effect of 1 microM vincristine (VCR) and inhibits glycerol-induced tubulin polymerization in a concentration-dependent manner (IC50: 125 microM), indicating that GaCl3 is a MT de-stabilizing agent that prevents MT assembly. However, 150 microM GaCl3 must be used to match or surpass the inhibitions of tubulin polymerization caused by 0.25 microM of known MT de-stabilizing agents, such as colchicine (CLC), nocodazole, podophyllotoxin, tubulozole-C and VCR. The inhibitory effect of 250 microM GaCl3 persists in the presence of up to 9 mM MgCl2, suggesting that the exogenous Mg2+ cations absolutely required for the binding of GTP to tubulin and MT assembly cannot overcome the antitubulin action of Ga3+ ions of a higher valence. The binding of [3H]vinblastine (VBL) to tubulin (0.5 mg/ml) is inhibited by unlabeled VBL but enhanced by concentrations of GaCl3 > 200 microM. However, increasing concentrations of GaCl3 mimic the ability of cold CLC to reduce the amount of [3H]CLC bound to tubulin, suggesting that GaCl3 may interact with the CLC binding site to inhibit tubulin polymerization. The binding of [3H]GTP to tubulin is decreased by unlabeled GTP but markedly enhanced by GaCl3, especially when concentrations of this metal salt of 32 microM or higher are added to the reaction mixture before rather than after the radiolabeled nucleotide. These data suggest that changes in protein conformation following GaCl3 binding might increase the interactions of tubulin with nucleotides and Vinca alkaloids. After a 24 h delay, the viability of GaCl3-treated L1210 leukemic cells is reduced in a concentration-dependent manner at days 2 (IC50: 175 microM), 3 (IC50: 35 microM) and 4 (IC50: 16 microM). Since GaCl3 (100-625 microM) increases the percentage of mitotic cells at 2-4 days, it might arrest tumor cell progression in M phase, but its antimitotic activity is much weaker than that of 0.25 microM VCR. Because the concentrations of GaCl3 that inhibit tubulin polymerization also increase the mitotic index and decrease the viability of L1210 cells in vitro, the antitubulin and antimitotic effects of GaCl3 might contribute, at least in part, to its antitumor activity.     

Inhibition of tubulin polymerization by vitilevuamide, a bicyclic marine peptide, at a site distinct from colchicine, the vinca alkaloids, and dolastatin 10

Vitilevuamide, a bicyclic 13 amino acid peptide, was isolated from two marine ascidians, Didemnum cuculiferum and Polysyncranton lithostrotum. Vitilevuamide was cytotoxic in several human tumor cell lines, with LC(50) values ranging from 6 to 311nM, and analysis in a 25-cell line panel revealed a weak correlation with several taxol analogs. Vitilevuamide was strongly positive in a cell-based screen for inhibitors of tubulin polymerization. Vitilevuamide at 9 microg/mL (5.6 microM) had an effect equivalent to the maximal effect of colchicine at 25 microg/mL (62.5 microM). Vitilevuamide was active in vivo against P388 lymphocytic leukemia, increasing the lifespan of leukemic mice 70% at 30 microg/kg. We hypothesized that at least part of the cytotoxic mechanism of vitilevuamide was due to its inhibition of tubulin polymerization. Vitilevuamide was found to inhibit polymerization of purified tubulin in vitro, with an IC(50) value of approximately 2 microM. Cell cycle analysis showed that vitilevuamide arrested cells in the G(2)/M phase with 78% of treated cells tetraploid after 16hr. Therefore, vitilevuamide was tested for its ability to inhibit binding of known tubulin ligands. Vitilevuamide exhibited non-competitive inhibition of vinblastine binding to tubulin. Colchicine binding to tubulin was stabilized in the presence of vitilevuamide in a fashion similar to vinblastine. Dolastatin 10 binding was unaffected by vitilevuamide at low concentrations, but inhibited at higher ones. GTP binding was also found to be weakly affected by the presence of vitilevuamide. These results suggest the possibility that vitilevuamide inhibits tubulin polymerization via an interaction at a unique site.     

Propylene glycol-mediated cell injury in a primary culture of human proximal tubule cells.

Propylene glycol (propane-1,2-diol; PD) is a widely used solvent for intravenous drugs. Clinical studies have reported serious side effects, including the development of renal insufficiency in patients receiving PD as drug vehicle. Despite such clinical reports, the data on the toxicity of PD in isolated renal cells are limited. Using primary cultured human proximal tubule (HPT) cells as an in vitro model, we have previously shown the acute toxic effects of PD in HPT cells (Morshed et al., Fundam. Appl. Toxicol. 23, 38-43, 1994). Since most cases of clinical toxicity are noted after prolonged administration of PD, the current studies were designed to investigate the toxicity of repeated exposure of PD in HPT cells. The onset of toxicity was determined using 10-50 mM racemic, sinister, and rectus PD (rac-, S-, and R-PD, respectively) for periods up to 6 days. Cytotoxicity was noted by decreases in thymidine incorporation, in mitochondrial metabolic activity, and in lysosomal accumulation of neutral red. Exposure of HPT cells to 50 mM PD produced toxic responses, while at 10 mM, responses were not significantly greater than those of osmotic controls. The toxicity was caused by a PD-specific mechanism and by a secondary mechanism without any enantiomeric specificity. The HPT cell toxicity was associated with a 35% increase in cellular thiobarbituric acid-reactive substances and a 20% decrease in glutathione. These findings suggest the development of a mild, subacute toxicity in normally proliferating HPT cells at concentrations that could be achieved in human plasma when PD is used as a drug vehicle.     

Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes.

Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.     

A comparison of BGM and LLC-PK1 cells for the evaluation of nephrotoxicity

Nephrotoxicity is one of the most frequent effects observed after the use of medicine. Such situations have been tardily discovered because of existing methods to determine toxicity. The validation of sensitive, alternative methods for the early identification of toxic effects is as important as restrictions on the use of animals. In this light, the present study evaluated the effects of gentamicin on BGM and LLC-PK1 cells, using MTT and Neutral Red (NR). Although the LLC-PK1 cell line is used for toxicological studies, the BGM cell line is relatively new for this purpose. MTT (BGM: EC(50)?=?6.29 mM; LLC-PK1: EC(50)?=?8.01 mM) was found to be more sensitive than NR (EC(50) was greater than 10 mM for both cells). By using MTT, both cells demonstrated the involvement of mitochondria in a manner that was dose dependent, with an apoptotic process occurring at the concentrations of 1 and 3 mM and necrosis at concentrations above 4 mM. It could, therefore, be concluded that 1) BGM appears to be useful in the study of the mechanism of nephrotoxicity caused by gentamicin and 2) because of its sensitivity to MTT, in addition to its ease of manipulation, it is believed that the BGM cell line can also be used as an alternative method to evaluate nephrotoxicity.     

Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia. Interaction with tubulin and effects of cellular microtubules.

Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. We have compared dolastatin 15 with dolastatin 10 for its effects on cells grown in culture and on biochemical properties of tubulin. The IC50 values for cell growth were obtained for dolastatin 15 with L1210 murine leukemia cells, human Burkitt lymphoma cells, and Chinese hamster ovary (CHO) cells (3, 3, and 5 nM with the three cell lines, respectively). For dolastatin 10, IC50 values of 0.4 and 0.5 nM were obtained with the L1210 and CHO cells, respectively. At toxic concentrations dolastatin 15 caused the leukemia and lymphoma cells to arrest in mitosis. In the CHO cells both dolastatin 15 and dolastatin 10 caused moderate loss of microtubules at the IC50 values and complete disappearance of microtubules at concentrations 10-fold higher. Despite its potency and the loss of microtubules in treated cells, the interaction of dolastatin 15 with tubulin in vitro was weak. Its IC50 value for inhibition of glutamate-induced polymerization of tubulin was 23 microM, as compared to values of 1.2 microM for dolastatin 10 and 1.5 microM for vinblastine. Dolastatin 10 noncompetitively inhibits the binding of vincristine to tubulin, inhibits nucleotide exchange, stabilizes the colchicine binding activity of tubulin, and inhibits tubulin-dependent GTP hydrolysis (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990; Bai et al. J Biol Chem 265: 17141-17149, 1990). Only the latter reaction was inhibited by dolastatin 15. Nevertheless, its structural similarity to dolastatin 10 indicates that dolastatin 15 may bind weakly in the "vinca domain" of tubulin (a region of the protein we postulate to be physically close to but not identical with the specific binding site of vinca alkaloids and maytansinoids), presumably in the same site as dolastatin 10 (the "peptide site").     

In vitro studies on the toxicity of isoniazid in different cell lines

The aim of the present study was to investigate in vitro the mechanism of toxicity of isoniazid (= INH), the drug most widely used for treatment of tuberculosis. The human hepatoma line HepG2, the human lymphoblastoid line AHH-1 and the murine lymphoma cells YAC-1 were used as test systems. Active cell death (= apoptosis) and necrosis were detected by different flow cytometric methods: the binding of annexin V to the cell membrane and staining with propidium iodide (PI), the TUNEL assay for detection of DNA fragmentation and the occurrence of a sub G1 peak in cell cycle histograms. Mitochondrial membrane potential was analysed with the fluorescent probe JC-1. In addition to cytotoxicity, effects of INH on cell cycle were studied in HepG2 cells. The data of the present investigations indicate that INH induces cytotoxicity via apoptosis both in hepatoma and lymphoma cells. Twenty-four hours of application of INH in concentrations > 26 mM led to a remarkable number of apoptotic cells positive for Annexin V. The induction of apoptosis was accompanied by a break down of the mitochondrial membrane potential and the occurrence of DNA strand breaks. At incubation times from 36 to 48 hours, a sub-G1 peak of late apoptotic cells was detected in cell cycle analysis. Furthermore, cell cycle studies showed a disruption of the cycle at low concentrations of INH which are only mildly cytotoxic. Thus the present study unequivocally demonstrated that INH induces cytotoxicity via apoptosis and can lead to a significant disturbance of the cell cycle in mammalian cells.