IC20 Values Research Articles

Squamous Cell Cancer Cell Lines: Sensitivity to Bleomycin and Suitability for Animal Xenograft Studies

Bleomycin (BLM) is a natural antibiotic, toxic to dividing cells (G2/M-phase), also proven effective in squamous cell carcinomas (SCC). We have clinically shown that a short-range β-emitting radionuclide combined to bleomycin (In-III-BLMC) is a tumor-targeting agent in SCCs. With higher radionuclide activities it may be possible to develop a more effective agent, to be tested in animal studies. Using a 96-well clonogenic assay we investigated three SCC cell lines, grown in our own laboratory. IC20, IC50 and IC90 values for BLM were determined. The UT-SCC-12A and UT-SCC-12B cells were originated from a primary tumor and a metastasis of the same patient. UT-SCC-12A cells were also inoculated subcutaneously into nude mice and the tumor growth was analysed. The IC50 value for UT-SCC-19A cell line was 4.0 ± 1.3 nM. UT-SCC-12A and UT-SCC-12B were both more resistant to BLM; IC50 values were 14.2 ± 2.8 nM and 13.0 ± 1.1 nM, respectively. Within 35 days the weight of nude mice increased 2.8 ± 0.6g. At 25 and 35 days after tumor inoculations the tumor volumes were 111 ± 51 mm3 and 874 ± 577 mm3, respectively. The calculated doubling time was 3.86 ± 0.76 days. SCC cell lines demonstrate different sensitivity to BLM. Our SCC tumor xenograft model showed a rapid growth proper for radiochemotherapeutic studies using In-III-BLMC. The uptake of In-111-BLMC in vivo has been directly proportional to proliferation activity, and the tumors with high binding capacity could be predicted from animal model dose calculations.

Calcium dynamics in cardiac myocytes as a target of dichloromethane cardiotoxicity

The purpose of the present study was to determine if cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca2+ dynamics in cardiac myocytes. Neonatal rat ventricular myocytes were obtained from 2- to 4-day-old rats, and electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were investigated using spectrofluorometric analysis of fura-2-[Ca2+]i binding. In cultured myocytes, cumulative exposure to 0.64-40.96 mM DCM resulted in a concentration-dependent and reversible decrease in the magnitude of [Ca2+]i transients with IC10 and IC50 values of 7.98 and 18.82 mM, respectively. Total inhibition of [Ca2+]i transients and cessation of beating were observed at 40.96 mM DCM. Suffusion with DCM for 40 min did not cause morphological alterations of the myocytes. In a urethane-anesthetized rat model, left ventricular pressure was measured by introducing a tip catheter via the carotid artery into the left ventricle, the ECG was recorded by two needle electrodes applied subcutaneously to the chest wall, and arterial pressure was measured via the femoral artery. Oral administration of 3.1-12.4 mmol DCM/kg resulted in DCM blood concentrations between 1.0 and 1.6 mM, accompanied by a dose-dependent decrease in contractile force and heart rate without influencing blood pressure and ECG tracings. Moreover, DCM treatment provided significant protection against arrhythmia development due to CaCl2-infusion. In spite of the slight discrepancy between DCM blood concentrations and in vitro concentrations of DCM for [Ca2+]i transient inhibition, present data are consistent with the view that cardiac effects after DCM exposure are mediated by alterations of Ca2+ dynamics during excitation-contraction coupling.

Determination of Critical Cellular Neurotoxic Concentrations in Human Neuroblastoma (SH-SY5Y) Cell Cultures

The effects of the neurotoxic compounds acrylamide, triethyltin chloride (TET), methyl mercury (II) chloride (MeHg) and lindane on selected neurospecific and general cell functions in differentiated human neuroblastoma SH-SY5Y cells were investigated in an attempt to determine critical cellular neurotoxic concentrations. The cultures were exposed to the neurotoxicants for three days, and then the effects on cell growth, neuronal signal transaction and the induction of axonopathy were measured. For comparison, general cytotoxicity was also determined in human epithelial (HeLa) cells. The cytotoxic activities (IC20 values) in the SH-SY5Y cells were 0.18 ± 0.03μM for TET, 0.20 ± 0.03μM for MeHg, 32 ± 10μM for lindane and 810 ± 170μM for acrylamide. Inhibition of cell growth was similar in HeLa cells. Significantly lower concentrations of MeHg, acrylamide and TET than the IC20 values were sufficient to induce axonopathy. In addition, MeHg and acrylamide increased the basal intracellular free calcium concentration, [Ca2+]i,as well as the carbachol-induced Ca2+fluxes and the depolarisation-stimulated increase of [Ca2+]icompared to control cells. The elevated [Ca2+]imay be a primary cause of the acrylamide-induced and MeHg-induced neuropathy. Treatment with lindane (1μM) slightly decreased the depolarisation-evoked Ca2+influx in the neuroblastoma cells. A parallel to the documented neurotoxic mechanism of lindane (i.e. inhibition of the γ-aminobutyric acid-activated Cl-channels) is possible.

Characterization of a human bladder cancer cell line selected for resistance to BMY 25067, a novel analogue of mitomycin C.

This study describes characteristics of a human bladder cancer cell line, SCaBER/R, selected for resistance to a mitomycin C (MMC) analogue BMY 25067. The SCaBER/R cell line was isolated by repeated 24 h exposures of the parental cells to 0.09 microM BMY 25067 (IC90, 24 h drug exposure) over a period of about 180 days. Approximately 2.2-fold higher concentration of BMY 25067 was required to kill 50% of the SCaBER/R cell line compared with parental cells (p < 0.001). The IC20 and IC90 values for BMY 25067 were also significantly higher in the SCaBER/R cell line than in SCaBER. Unlike most MMC resistant cell lines, the SCaBER/R cell line displayed a marked cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and lacked cross-resistance to cisplatin, doxorubicin or VP-16. The SCaBER/R cell line also displayed a marked cross-resistance to the parent drug (MMC) and BMY 25282, another analogue of MMC. NADPH cytochrome P450 reductase activity, an enzyme implicated in bio-reductive activation of MMC, did not differ significantly in these cells. DT-diaphorase activity, another MMC activation enzyme, was significantly lower in the SCaBER/R cell line when compared to the SCaBER cells. These results suggest that relatively lower sensitivity of SCaBER/R cell line to MMC and BMY 25067 may result from impaired drug activation. Cellular levels of glutathione (GSH) and GSH-transferase (GST), which have been suggested to affect the cytotoxicity of MMC, were comparable in SCaBER and SCaBER/R cell lines. BMY 25067 induced DNA interstrand cross-links (DNA-ISC) could not be detected in either of the cell lines even at drug concentrations which produced a significant cell kill. These findings suggest that (a) cellular resistance to BMY 25067 in the SCaBER/R cell line may be due to impaired drug activation, and (b) the nature of the cytotoxic produced by BMY 25067 may be different from that of MMC.

In vitro activity of S 9788 on a multidrug-resistant leukemic cell line and on normal hematopoietic cells?reversal of multidrug resistance by sera from phase I-treated patients

The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials. In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype. S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs. At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells. S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors. The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied. Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788. In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration. In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells. The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM. These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

Toxic Effects of Pollutants on the Mineralization of Chloroform in River Sediments

The influence of pollutants on the formation of 14CO2 from 3 μg/liter labeled chloroform was studied in anaerobic Dutch river sediments. All incubations were performed under anaerobic conditions. Addition of toxicants to sediment microcosms showed logistic dose-effect curves. The concentration giving 10% inhibition of the chloroform mineralization rate (IC10) was derived from these dose-effect curves. The IC10 values of added cadmium, chloropyrifos, benzene, mercury, or 1,2-dichloroethane were 1300, 1300, 140, 90, and 0.07 mg/kg dry sediment, respectively. Mud samples taken at different dates from the same site indicated a significantly different sensitivity to added pentachlorophenol and zinc. The IC10 of added pentachlorophenol was 150 mg/kg in one and 15 mg/kg in another sample. Chloroform-mineralizing bacteria are very sensitive to addition of zinc. The IC10 of added zinc was 700 mg/kg for one sample and 11 mg/kg for another sample of the sediment which contained a background concentration of 800 mg Zn/kg. Therefore, a partial inhibition of the mineralization of chloroform by the high concentrations of zinc present in Dutch river sediments cannot be excluded. The high concentration of zinc might cause persistence of otherwise biodegradable pollutants in Dutch sediments.

N-methyl-D-aspartate (NMDA)-stimulated noradrenaline (NA) release in rat brain cortex is modulated by presynaptic H3-receptors.

In superfused rat brain cortex slices and synaptosomes preincubated with [3H]noradrenaline the effect of agonists or antagonists at presynaptic H3 receptors on NMDA-evoked [3H]noradrenaline release was investigated. In experiments on slices, histamine and the preferential H3 receptor agonist R-(-)-alpha-methylhistamine inhibited NMDA-evoked tritium overflow (IC20 values 0.27 mumol/l or 0.032 mumol/l, respectively); S-(+)-alpha-methylhistamine (up to 10 mumol/l) as well as the selective H1 receptor agonist (2-(2-thiazolyl)ethylamine and the selective H2 receptor agonist dimaprit (each up to 10 mumol/l) were ineffective. The H3 receptor antagonist thioperamide abolished the inhibitory effect of histamine whereas the preferential H1 receptor antagonist dimetindene and the preferential H2 receptor antagonist ranitidine were ineffective. In experiments on synaptosomes, histamine and R-(-)-alpha-methylhistamine inhibited NMDA-evoked tritium overflow, whereas 2-(2-thiazolyl)ethylamine or dimaprit had no effect. The inhibitory effect of histamine was abolished by thioperamide. When tritium overflow was stimulated by NMDA in the presence of omega-conotoxin GVIA (which by itself decreased the response to NMDA by about 55%), R-(-)-alpha-methylhistamine did not inhibit NMDA-evoked overflow. It is concluded that NMDA-evoked noradrenaline release in the cerebral cortex can be modulated by inhibitory H3 receptors. NMDA receptors and H3 receptors are both located presynaptically and may interact at the same noradrenergic varicosity. An unimpaired function of the N-type voltage-sensitive calcium channel probably is a prerequisite for the inhibition of NMDA-evoked noradrenaline release by H3 receptor stimulation.

Evaluation of the Cytotoxicity of the First Ten MEIC Chemicals in 3T3 Mouse Fibroblasts with and without Microsomal Activation, Using the Neutral Red Assay

The cytotoxicity of the first ten MEIC chemicals was evaluated by determination of neutral red uptake in BALB/C 3T3 mouse fibroblasts both with and without the addition of a microsomal activation system. The IC20 values of all the chemicals tested without microsomes were significantly correlated to in vivo data on acute systemic toxicity. The use of microsomes weakened the overall correlation with the in vivo data. Useful information regarding the activation of acetylsalicylic acid, iron sulphate, methanol and ethanol was obtained using microsomes, while misleading information was obtained regarding the metabolism of paracetamol, diazepam, digoxin and ethylene glycol. In the case of alcohol metabolism, the Aroclor 1254 induction of rat liver enzymes seems to parallel the induction of cytochrome P450 IIE1, which is known to metabolise a number of alcohols to free radicals which might exert a variety of cytotoxic effects. For this reason, the use of Aroclor 1254 induction in cytotoxicity and genotoxicity testing is questioned.

Neurochemical studies on quinolone antibiotics: effects on glutamate, GABA and adenosine systems in mammalian CNS.

Quinolone antibiotics, which can be proconvulsant in susceptible patients, were found to inhibit the specific binding of the adenosine receptor ligands L-3H-N6-phenylisopropyladenosine (L-3H-PIA) and 3H-N-ethylcarboxamidoadenosine (3H-NECA) to rat brain synaptic membranes. The inhibitions were concentration dependent, and for both ligands the order of potency was rosoxacin greater than nalidixic acid greater than oxolinic acid greater than or equal to ciprofloxacin greater than norfloxacin greater than enoxacin: IC20 values (concentrations causing a 20% inhibition of specific binding) ranged from 30-35 microM to 1-3 mM. Hill coefficients were approximately 0.5, suggesting that the compounds are probably antagonists at these sites. Most of the compounds did not alter 3H-diazepam binding directly, although rosoxacin showed relatively strong, and enoxacin weak, concentration-dependent inhibition. At 50 microM the compounds enhanced the maximal gamma-aminobutyric acid (GABA) activation of 3H-diazepam binding to varying degrees, without altering the EC50 of activation, whereas at 200 microM they tended to reduce GABA activation. Most noteworthy was the large increase in GABA-stimulated 3H-diazepam binding caused by 50 microM nalidixic acid. The compounds did not alter the Ca2+/Cl- -dependent binding of 3H-glutamate, nor of the binding of the glutamate site-selective ligands 3H-kainate and alpha-3H-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA); the uptake of the non-metabolized glutamate analogue D-3H-aspartate by cortical homogenates was also unaffected. The CNS side effects of these antibiotics may result, in part, from interaction with sites which mediate the inhibitory neurotransmission of adenosine and, possibly, GABA.

Validity of an Ecotoxicological Test System: Short-Term and Long-Term Effects of Arsenate on Marine Periphyton Communities in Laboratory Systems

Periphyton communities were established on glass substratum in the Gullmar Fjord (Sweden) and their sensitivity to arsenate was determined with short-term photosynthesis experiments. The study was done in May 1984 when phosphate levels were low. A 1-h exposure to arsenate gave IC20 (concentration required to inhibit photosynthesis by 20%) values of 0.4 μM. Communities established indoors in a flow-through aquaria system were substantially different from the fjord communities in terms of structure and biomass, although photosynthesis showed a similar sensitivity (IC20 = 0.6 μM). To explore the validity of this short-term ecotoxicological test system, we used a set of indoor, flow-through aquaria to study long-term effects (3 wk) on communities established under arsenate stress. Arsenate was added continuously at 0.1–10 μM above background levels (7 nM). Species composition as well as carbon, nitrogen, and chlorophyll a content of the communities were affected at 0.2–0.8 μM (IC20). This is in good agreement with the sensitivity of the short-term test. We conclude that inhibition of the physiological parameter photosynthesis is relevant information for the understanding of ecological events caused by arsenate stress in the periphyton communities. At least for effects of arsenate, the short-term metabolic test system can be used for valid predictions of gross changes in periphyton community structure and production. Comparison with arsenic levels in polluted coastal areas of Sweden lead to the conclusion that periphyton communities may be affected, particularly when phosphorus levels are low.

Decreased endothelium-dependent relaxation in New Zealand genetic hypertensive rats.

The relaxation response to endothelium-dependent (acetylcholine and the calcium ionophore A23187) and independent (sodium nitroprusside) vasodilators was examined in isolated aortic ring segments from age-matched genetically hypertensive (GH) and normotensive (N) rats (New Zealand strain). Tissues were initially contracted with methoxamine to achieve similar levels of contractile force. The IC20, IC40 and IC50 values for acetylcholine, A23187 and sodium nitroprusside were shifted significantly to the right (P less than 0.05) in aortic rings from GH rats compared to the corresponding values in N rats. The maximal relaxation achieved by acetylcholine and A23187 was significantly depressed in aortas from GH rats (P less than 0.05). Sodium nitroprusside elicited the maximal relaxation in both groups of tissues. These results demonstrate that there exists a generalized defect in the relaxant ability of vascular smooth muscle from GH rats. In addition, our findings suggest that this defect is coupled with a decreased responsiveness to endothelium-dependent vasodilators in this particular animal model of hypertension.