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PubMed2006

Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.

Caspase 3↗
PubMed2002

Apoptotic chondrocyte death in cell-matrix biocomposites used in autologous chondrocyte transplantation.

Tissue engineering may be a promising approach for the treatment of focal articular cartilage defects. Programmed cell death (apoptosis) plays an important role in multiple degenerative processes of cartilage (e.g. osteoarthritis). It is known that matrix provides a trophic signal for the cells and an altered matrix may influence the availability of factors that regulate apoptosis. In this study we investigate the viability of chondrocytes seeded on a Chondrogide scaffold (Geistlich Biomaterials, CH), which we use in matrix-induced autologous chondrocyte transplantation (MACT). By now, we have studied material from 29 patients treated for localized articular cartilage defects in the knee. Our results indicate that light microscopy (Mayer's hematoxylin-eosin, Masson-Goldner, Trypan-blue and TUNEL method) and electron microscopy can be used to investigate for apoptotic cells grown on a Chondrogide resorbable scaffold. Neither the handling of the cell-matrix biocomposite nor the procedures for fixation could destroy the scaffold or the cell sheet adhering firmly to the matrix. Apoptotic cells were revealed in all samples and with all techniques used. Mayer's hematoxylin-eosin and Masson-Goldner staining show cells with a condensed, pycnotic nucleus and shrunken cytoplasm. In electron microscopy we observed cells with chromatin condensation and volume shrinkage consistent with apoptosis. The results of the Trypan-blue staining show a mean viability of 92.1 +/- 9.8% (range 57-100%). The TUNEL method revealed 44.6 +/- 20.4% positive cells. Our results indicate that apoptosis plays an important role in chondrocytes grown on a scaffold. An optimal scaffold will determine the growth, morphology and phenotype of the chondrocytes by its physical and chemical characteristics.

Adult↗
PubMed1987

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ.

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.

Animals↗
PubMed2006

Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene.

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.

Animals↗
PubMed1991

Histological evidence for dissociation of lipid peroxidation and cell necrosis in bromotrichloromethane hepatotoxicity in the perfused rat liver.

Bromotrichloromethane (CBrCl3)-induced hepatic lipid peroxidation and cell necrosis were studied histologically and biochemically, using isolated perfused livers from phenobarbital-pretreated rats. Lipid peroxidation was assessed by fuchsin staining of the liver slices and release of thiobarbituric acid reactive substances (TBARS) into the perfusate; necrosis was assessed by trypan blue uptake and lactate dehydrogenase (LDH) leakage. A good correlation was observed between the Schiff-positive reaction and TBARS release under various experimental conditions, supporting the validity of the fuchsin staining method for histological detection of lipid peroxidation. Lobular localization of lipid peroxidation and necrosis was as follows: Under high oxygen supply (95% O2-saturated buffer), infusion of CBrCl3 caused the Schiff-positive reaction in the pericentral to midzonal hepatocytes, irrespective of the direction of perfusion, but did not produce necrosis. Under low oxygen supply (20% O2) with retrograde perfusion, dissociation of lipid peroxidation and necrosis was observed, i.e., trypan blue uptake in the periportal zones and Schiff-positive staining in the pericentral hepatocytes. Thus, lipid peroxidation by itself may have a relatively minor role in the development of CBrCl3-induced acute hepatic cell death.

Animals↗
PubMed2005

Assessment of DNA damage in target tumor cells after thermoablation in mice.

PURPOSE: To determine the effects of temperature on cell death for cells in culture and to compare these effects with the results of in vivo experiments in which heating is induced in mice with implanted human adenocarcinoma by using magnetic methods. MATERIALS AND METHODS: Experimentation was approved by the regional animal care committee. Human adenocarcinoma cells (MX-1) and human fibroblasts (HTB-125) were exposed to defined temperatures of 45 degrees -90 degrees C for 4 minutes. Single- and double-strand DNA breaks (expressed as a percentage of the total DNA in tail) were identified by using the alkaline comet assay, and cell survival was determined by using the cloning assay and trypan blue exclusion. For in vivo experiments, MX-1 tumors were implanted into 14 mice. Magnetic heating at temperatures of 59 degrees -96 degrees C was subsequently performed by injecting magnetic material into the tumor (7 mg +/- 3 [+/- standard deviation] magnetite per tumor) and applying an alternating magnetic field (8.8 kA/m; 400 kHz) for 4 minutes. The efficiency of the temperature-dependent induction of DNA damage in isolated tumor cells was quantified and compared with that in cultured cells. RESULTS: Results of experiments with cell cultures revealed a strong correlation between DNA damage, cell survival, and temperature, as determined with the cloning assay and trypan blue exclusion. The threshold thermoablasive temperature for tumor cell elimination was found to be 55 degrees-60 degrees C. Moreover, a strong impairment in cell survival was found when damaged DNA accounted for more than 50% of the total DNA. The heating sensitivities of malignant and nonmalignant cells did not differ. After the magnetic heating of tumors in vivo to temperatures of up to 96 degrees C (rectal temperatures between 27 degrees C +/- 2 and 29 degrees C +/- 2), isolated tumor cells showed a mean of 71.9% +/- 24.5 of total DNA in the tail per cell compared with nontreated tumors, which showed 8.0% +/- 3.1. CONCLUSION: There appears to be a threshold temperature for the induction of irreversible DNA damage. This is reflected by the results of in vivo experiments in tumor-bearing mice after high temperatures were applied to tumors by using magnetic heating.

Adenocarcinoma↗
PubMed1988

Effects of SO2 or NOx on toxic and genotoxic properties of chemical carcinogens. II. Short term in vivo studies.

Short term in vivo studies were performed to study biological effects of the common air pollutants SO2 or NOx and their influence on the genotoxic activities of nitrosamines. Hepatocytes and lung cells were isolated from Sprague-Dawley rats which had inhaled 50 p.p.m. of SO2 or NOx for 2 weeks. After incubating the cells for 1 h, genotoxicity was determined in hepatocytes by measuring DNA single-strand breaks induced by N-nitroso-acetoxymethylmethylamine, N-nitrosodimethylamine and N-nitrosomethylbenzylamine. Parameters of toxicity (trypan blue exclusion and leakage of serum enzymes) were determined in both liver and lung cells also following 1 h incubation. The activities of aryl hydrocarbon hydroxylase (AHH), nitrosodimethylamine demethylase (NDMA-D) and glutathione-S-transferase (GST) were determined in subcellular microsomal fractions isolated from lung and liver tissues. Finally, as a measure of overall toxicity, the activities of various serum enzymes were determined in the blood serum of the rats. It was found that the induction of DNA single-strand breaks by three nitrosamines was decreased in hepatocytes from SO2-treated animals. The viability of rat hepatocytes and of rat lung cells, as determined by trypan blue exclusion, was similar in all three treatment groups immediately after isolation, as well as after 1 h incubation with DMSO or with the nitrosamines. In contrast, the leakage of enzymes was different in hepatocytes of SO2-treated rats, since lactate dehydrogenase activity was decreased. Leakage of enzymes from the lung cells did not differ from group to group, but was lower than from hepatocytes. Foreign compound metabolizing enzymes were mainly decreased in NOx-treated animals, namely AHH, NDMA-D and GST in liver and GST in the lung. For SO2-treated animals NDMA-D was increased in liver and GST was decreased in lung. Blood serum enzyme levels were not greatly different from each other, except for lactate dehydrogenase which was elevated in SO2-exposed animals.

Air Pollutants↗
PubMed1991

Some antagonists of platelet activating factor are cytotoxic for human malignant cell lines.

Nine new platelet activating factor (PAF) antagonists from 4 different chemical classes (thiopyrimidines: SDZ 59-015; thioimidazolines: SDZ 61-813; imidazoisoquinolines: SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596; and imidazopiperidines: SDZ 61-638, SDZ 62-293, SDZ 62-694) have been tested for cytostatic/antiproliferative ([3H]thymidine uptake) and cytotoxic (trypan blue dye exclusion) activity in neoplastic human cell lines of different histology in vitro. The antiproliferative activity of 3 of the 9 PAF antagonists (SDZ 61-638, SDZ 61-813, SDZ 62-694) was not stable after freezing and thawing. SDZ 59-015 showed only minor cytotoxic or antiproliferative effects in a dose range of 2-40 microns after 24, 48, and 72 h of incubation. SDZ 62-434 showed varying activity. There were no significant differences between the activities of the other 3 PAF antagonists from the imidazoisoquinoline class, which showed drug concentrations inhibiting 50% of the activity studied (IC50) and drug concentrations yielding a 50% decrease of trypan blue dye exclusion (LC50) of less than or equal to 20 microM at greater than or equal to 48 h, even in the K-562 cell line, which is known to be rather resistant for a variety of cytotoxic drugs related to PAF. SDZ 62-293 showed the best antineoplastic properties with IC50 and LC50 values less than or equal to 10 microM at greater than or equal to 48 h including K-562. SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596, and SDZ 62-293 have been further tested in a human tumor cloning assay in 5 cell lines. Colony formation was reproducibly suppressed to less than 30% of the controls only by SDZ 63-135 (less than or equal to 40 microM) and SDZ 62-293 (less than or equal to 20 microM) during continuous exposure. There was no correlation between the IC50 values for the antiproliferative activity of the test compounds and their IC50 values for PAF-induced human platelet aggregation. Furthermore, the antiproliferative activity of the most active compound, SDZ 62-293, could not be antagonized by preincubation with the specific PAF antagonists WEB 2170 or WEB 2086 or PAF itself in noncytotoxic doses.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗
PubMed1989

Assessment of the viability of haemopoietic cells in the livers of mouse fetuses stored at 4 degrees C. Comparison of various methods of testing their usefulness for haemopoietic transplantation.

The usefulness of three different tests available in experimental haematology for the assessment of the viability of fetal liver cells obtained from murine fetuses kept in refrigerator at 4 degrees C was compared. The usefulness of these cells as potential transplants for haemopoiesis reconstitution was assessed in the test with trypan blue, in the test based on clonal growth of GM-CFU in agar, and in the test of the splenic colony forming ability of CFU-S. Fetuses kept in refrigerator at 4 degrees C tested 16 hours after circulation arrest contained still about 70% of the initial number of the haemopoietic stem cells (CFU-S). The proportion of committed cells belonging to the granulocyto-monopoiesis line (GM-CFU) decreased at the same time to about 25% of the initial number, and was a sensitive indicator of hypoxia of the studied organ. The test for the viability of the cells based on the use of trypan blue gave results reflecting better the changes in the number of CFU-S than GM-CFU cells.

Animals↗
PubMed2005

[Collection of peripheral blood stem cells from ABO incompatible allogeneic donors by using blood cell separator].

To evaluate the yield of the blood cell separator for collection of peripheral blood stem cells (PBSC) from ABO major and (or) minor incompatible allogeneic donors and the feasibility of PBSC component infusion to the recipients without removal of erythrocytes or plasma, the Cobe Spectra (Version 6.1) blood cell separator was utilized to collect PBSC component from 9 allogeneic donors. Of all the donors, 4 were ABO major incompatible, 2 were minor incompatible and the other 3 were both major and minor incompatible to corresponding recipients. In each cycle, different amount of PBSC component was harvested, and the variable volume plasma chased the cells into the bag was adjusted according to the ABO incompatibility. The nucleated cell count, percentage of mononuclear cell, number of CD34(+) cell and percentage of viable cell (trypan blue excluding rate) in the component were detected. At the time of infusion, a series of protective measures to the renal function of recipients were taken. The results showed that apheresis was twice performed on these eight donors to collected enough PBSC for transplantation or cryopreservation, except one apheresis was enough for cell amount needed by transplantation, as the donor's body weight was much heavier than that of the recipient. Altogether 17 apheresises were performed, the mean yield of nucleated cells was 3.77 x 10(10), in which 97% to 99% were mononuclear cells (MNC). The harvested number of CD34(+) cell was 8.62 x 10(6)/kg. All the trypan blue exclusion rate was 100%. In ABO major incompatible or both major and minor incompatible component, there were 8 - 10 ml packed erythrocytes; in ABO minor incompatible component, there were 80 - 120 ml of plasma. These components were infused into the recipients without removal of erythrocytes or plasma and no haemolytic reaction was observed in any recipient, and their hematopoietic functions soon recovered. Results suggest that enough PBSC can be acquired by using blood cell separator Cobe Spectra (Version 6.1), with the modified separation factors, and the collected PBSC component can be safely infused into the ABO incompatible recipients without removal of erythrocytes or plasma.

ABO Blood-Group System↗
PubMed2003

Lethiferous effects of a recombinant vector carrying thymidine kinase suicide gene on 2.2.15 cells via a self-modulating mechanism.

AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15 cells and the possible self-modulating mechanism. METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus antisense-S (HBV-anti-S) gene, hepatitis C virus core (HCV-C) gene, internal ribosome entry site (IRES) element of HCV and TK gene into the eukaryotic vector pcDNA3, in which the expression of TK suicide gene was controlled by the HBV S gene transcription. 2.2.15 cells that carry the full HBV genome and stably express series of HBV antigen were transfected with pcDNA3-SCITK or vector pcDNA3-SCI which was used as the mock plasmid. The HepG2 cells transfected with pcDNA3-SCITK were functioned as the negative control. All the transfected cells were incubated in DMEM medium supplemented with 10 microg/ml. of ganciclovir (GCV). The HBsAg levels in the supernatant of cell culture were detected by ELISA on the 1st, 3rd and 6th day post-transfection. Meanwhile, the morphology of transfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th day post-transfection. RESULTS: The structural accuracy of pcDNA3-SCITK was confirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. The HBsAg levels in the supernatant of transfected 2.2.15 cell culture were significantly decreased on the 6th day post-transfection as compared with that of the mock control (P<0.05). The lethiferous effect of pcDNA3-SCITK expression on 2.2.15 cells was initially noted on the 3rd day after transfection and aggravated on the 6th day post transfection, in which the majority of transfected 2.2.15 cells were observed shrunken, round in shape and even dead. With assessment by the trypan blue exclusion test, the survival cell ratio on the 6th day post transfection was 95% in the negative control and only 11% in the experimental group. CONCLUSION: The results indicate that suicide gene expression of pcDNA3-SCITK can only respond to HBV-S gene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.

Cell Survival↗
PubMed2003

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion.

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44(MAPK)), the p38 kinase (p38(MAPK)), the c-Jun N-terminal kinases (p46/p54(JNK)), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/ p44(MAPK) as well as of Akt kinase was partially reduced. For p46/p54(JNK) and p38(MAPK), elevated phosphorylation was measured. Inhibition of p46/p54(JNK) and p38(MAPK) activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase 7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.

Animals↗
PubMed1980

Studies on the cytopathogenicity of Entamoeba histolytica.

We have explored the interaction of virulent and avirulent E. histolytica (HM1 and 303 respectively) with human polymorphonuclear leukocytes (PMNs) and Chinese hamster ovary (CHO) cells. PMNs exhibit chemotaxis to both HMl and 303. On contact with HMl, PMNs degranulate and die (trypan blue uptake); incubation at a ratio of 100 PMNs per ameba for 40 minutes revealed 70 per cent PMN death with 84 per cent HMl viability. 303s are often ingested by PMNs on contact; after incubation as above, only 1 per cent of 303 were intact with PMN mortality equaling that of control. HMl leukotoxicity occurred under anaerobic conditions, in immune serum, and was not mediated by complement. CHO cells are disrupted by HMls, less than 50 per cent of CHO monolayer remains after < 3 hours with 10(4) HMl/ml. Sonicated HMls (.5 x 10(5)/ml) washed in phosphate buffered saline caused cell release without cell death versus control (by trypan blue uptake). When 5 microgram/ml of Cytochalasin B was added to PMNs and CHO cells, cytopathogenicity by HMls decreased 62 per cent (p < .01) and nearly to control values (p < .01) respectively. Colchicine 0.25 mM had no significant effect on PMN cytopathogenicity. Washed HMl and 303 (1 x 10(5)/ml) were agglutinated by 10 microgram/ml and 100 microgram/ml of the lectin concanavalin A. Cytopathogenicity was associated with intact microfilament function and ameba motility. Surface membrane receptors may be important mediators of virulence. Further studies of potential membrane receptors and inhibitors are underway.

Animals↗
PubMed1994

Injuries to cultivated BJA-B cells by ajoene, a garlic-derived natural compound: cell viability, glutathione metabolism, and pools of acidic amino acids.

Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide), a garlic-derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma-derived cell line (BJA-B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the GSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism.

Amino Acids↗
PubMed2004

The ceramide analog, B13, induces apoptosis in prostate cancer cell lines and inhibits tumor growth in prostate cancer xenografts.

BACKGROUND: Apoptosis is a therapeutic target for the elimination of cancer cells. As elevations in ceramide levels induce apoptosis, there is much excitement about the use of agents that elevate ceramide levels as novel chemotherapeutic agents. Ceramidases are enzymes involved in degradation of ceramide and inhibition of ceramidase has been proposed as a mechanism to increase ceramide levels. This study provides the first insight into the effect of B13, an inhibitor of acid ceramidase, on human prostate cancer cell lines and xenografts. METHODS: Cell death was evaluated by the trypan blue assay; apoptosis by the Apo2.7 apoptosis assay; and glutathione levels by HPLC. Tumors were irradiated with a dose of 5 Gy of X-rays (250 kVp, 15 mA, 2 Gy/min) and tumor volume was measured during the course of the experiment. At the conclusion of the experiment, tumor weight was determined and the tumors were evaluated histologically. RESULTS: B13 is an inducer of cell death, by apoptosis, in cultured prostate cancer cells. LNCaP and PC3 cells have different responsiveness to the enantiomers of B13. In LNCaP cells, the R enantiomer of B13 (10 microM) was significantly more effective than the S enantiomer at inducing cell death as determined by the trypan blue assay, culminating in approximately 90% cell death at 48 hr. In contrast, the same concentration of B13S induced <20% cell death at 48 hr. In PC3 cells, the S enantiomer was a more effective inducer of cell death, culminating in approximately 30% cell death, relative to 14% for B13R in this model. Evaluation of induction of apoptosis by the Apo2.7 mitochondrial assay confirmed that this induction of cell death was by apoptosis. Concurrent with induction of apoptosis, glutathione levels drop in response to B13. Specifically, B13R caused a significant drop in glutathione levels in LNCaP cells, culminating in a reduction to 40% control values at 48 hr. In PC3 cells, in contrast, the drop in glutathione levels was more dramatic, culminating in a drop to 12% control values in response to B13S at 48 hr. The effects of B13R, however, were not significantly different from control values. In in vivo studies using a model of xenografted androgen-insensitive prostate cancer, B13 sensitized the tumors to the effects of radiation, resulting in a significant reduction in tumor volume and weight after treatment with the combination of B13 and radiation. Microscopic evaluation of the tumors indicated that apoptosis was the primary mechanism of this effect. CONCLUSIONS: Targeting ceramide pathways may be a novel treatment strategy for hormone refractory prostate cancer.

Amides↗
PubMed1997

The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the gamma- and delta-isomers.

The cytotoxic action of the delta- and gamma-isomers of hexachlorocyclohexane (HCH) as well as their ability to induce changes in intracellular Ca2+ homeostasis was studied in cultured rat cerebellar granule neurons. Changes in the free intracellular Ca2+ concentration ([Ca2+]i) related to Ca2+ influx and release from intracellular stores were investigated using, in addition to the physiological incubation medium, media without added Ca2+ (nominally Ca2+ free) or containing verapamil or dantrolene, drugs which block influx through voltage-gated Ca2+ channels and release from intracellular stores, respectively. Cytotoxicity was monitored using leakage of lactate dehydrogenase (LDH) and staining of damaged cells with either trypan blue or propidium iodide. In the latter case, when fluorescence microscopy was employed, undamaged cells were visualized using the vital stain calcein acetoxymethyl ester (calcein-AM). The delta-isomer of HCH was found to be more potent and active as a cytotoxic agent than the gamma-isomer (lindane) and it was demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cytotoxicity induced by delta-HCH. It was noticed that pronounced (50% increase in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<25%) cells with trypan blue. Both delta- and gamma-HCH induced an increase in [Ca2+]i which was most pronounced in case of delta-HCH. Analysis of the different mechanisms governing the increase in [Ca2+li using dantrolene, verapamil, and Ca2+-free medium revealed distinct differences between the two isomers with regard to the Ca2+ pools affected. Both isomers stimulated Ca2+ influx through voltage-gated Ca2+ channels but only the gamma-isomer affected a Ca2+-dependent, dantrolene-sensitive pool. On the other hand, delta-HCH affected mainly a Ca2+-independent dantrolene-insensitive pool. The finding that delta-HCH was more toxic than lindane may be correlated to the differences between the isomers with regard to the action on the different Ca2+ pools. Thus, delta-HCH affected primarily the dantrolene-insensitive Ca2+ pools which partly reflect influx. On the contrary, lindane had little effect on these Ca2+ pools but affected primarily dantrolene-sensitive intracellular Ca2+ stores. This may suggest that delta-HCH may exert its cytotoxic action by stimulating a large influx of Ca2+ possibly leading to release of Ca2+ from dantrolene-insensitive stores. In contrast, the toxic action of lindane may be primarily related to release of Ca2+ from the dantrolene-sensitive stores.

Animals↗
PubMed1997

N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2).

PURPOSE: Previous studies have established that chitosan hydrochloride and glutamate are potent absorption enhancers for large hydrophilic compounds across mucosal surfaces. However, these compounds lack solubility at neutral pH values. A partially quaternized and well-soluble derivative of chitosan, N-trimethyl chitosan chloride, was synthesized and the effects of this polymer on the transepithelial electrical resistance and permeability of intestinal epithelial cells were investigated in vitro. METHODS: N-trimethyl chitosan chloride was synthesized by reductive methylation and characterized with NMR. The effect of this polymer (1.0-2.5% w/v) on the transepithelial electrical resistance of intestinal epithelial cells, using Caco-2 cell monolayers, was investigated. Permeation of the hydrophilic model compounds [14C]-mannitol (MW 182.2), FITC-Dextran (MW 4400) and the peptide drug buserelin (MW 1299.5), in the presence of N-trimethyl chitosan chloride (1.5-2.5% w/v), was followed for 3 hours. The transport process of the fluorescent marker, FITC-Dextran 4400, across the cell monolayers was visualised with confocal laser scanning microscopy. Viability of the cells was checked with the trypan blue exclusion technique. RESULTS: N-trimethyl chitosan chloride was found to be a perfectly water-soluble, partially quaternized (about 12%) derivative of chitosan. This polymer (1.5-2.5% w/v) caused a pronounced and immediate reduction (25-85%) in the transepithelial electrical resistance of Caco-2 cells. Large increases in the transport rate of [14C]-mannitol (32-60 fold), FITC-Dextran 4400 (167-373 fold) and buserelin (28-73 fold) were demonstrated. Confocal laser scanning microscopy confirmed that N-trimethyl chitosan chloride opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds through the paracellular transport pathway. No deleterious effects to the cells could be demonstrated with trypan blue. CONCLUSIONS: The potential use of N-trimethyl chitosan chloride as an absorption enhancer across mucosal surfaces could be an important contribution towards the development of effective delivery systems for hydrophilic drugs.

Biological Transport↗
PubMed1997

Aminoglycoside-induced viability in cultured PtK1 proximal tubular cell lines.

The cell viability after aminoglycoside administration was evaluated by the determination of adenosine triphosphate (ATP) level and trypan blue exclusion of cultured PtK1 tubular cells. The assay in tubular cells was performed using luciferin-luciferase determined by a chemiluminator. The data are expressed as relative light units per milligram tissue protein. The standard curve of ATP showed a linearity from 0.5 to 2.5 mg/ml ATP as determined by the chemiluminator. The incubation time was steady for ATP production during 90 min after addition of ligand. The optimal incubation time was 60 min. The ATP production in PtK1 cells showed linearity within 10(3) to 5 x 10(6) cells in 200 microliters buffer. Although the magnitude of ATP production in Ptk1 cells was correlated with the cell viability as defined by trypan blue exclusion, the reproducibility of the former was better than that of the latter. The cell viability after application of aminoglycoside showed a decrement in a dose-dependent fashion. Our ATP assay is an excellent tool for determining the cell viability of renal proximal tubules in cases with tubular damage after administration of aminoglycoside antibiotics.

Adenosine Triphosphate↗