Complete Dose-response Curves Research Articles

Cannabinoid and Opioid Receptor Affinity and Modulation of Cancer-Related Signaling Pathways of Machaeriols and Machaeridiols from Machaerium Pers.

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A-D (1-4) and machaeridiol A-C (5-7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A-C (5-7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 μM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1-7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose-response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5-7 and their cross-talk potential has been discussed.

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity

Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction, and liver enzyme inhibition). Five concentrations per substance, aimed at covering the complete dose-response curve, were analyzed and 221 uniquely identified metabolites were measured, annotated, and allocated in 12 different metabolite classes such as amino acids, carbohydrates, energy metabolism, nucleobases, vitamins and cofactors, and diverse lipid classes. Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based, and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines.Graphical abstract

Dose Effect of Mesenchymal Stromal Cell Delivery Through Cardiopulmonary Bypass

BackgroundNeurologic impairments are a significant concern for survivors after pediatric cardiac surgery with cardiopulmonary bypass (CPB). We have previously shown that mesenchymal stromal cell (MSC) delivery through CPB has the potential to mitigate the effects of CPB on neural stem/progenitor cells. This study assessed the dose effects of MSCs. MethodsPiglets (n = 20) were randomly assigned to 1 of 4 groups: control, CPB, or CPB followed by MSC administration with low and high doses (10 × 106 and 100 × 106 cells per kilogram). We assessed acute dose effect on cell distribution, multiorgan functions, systemic inflammation, microglia activation, and neural stem/progenitor cell activities. ResultsBy magnetic resonance imaging, approximately 10 times more MSCs were detected within the entire brain after high-dose delivery than after low-dose delivery. No adverse events affecting hemodynamics, various biomarkers, and neuroimaging were detected after high-dose MSC delivery. High-dose MSCs significantly increased circulating levels of interleukin 4 after CPB. Both MSC groups normalized microglia activation after CPB, demonstrating MSC-induced reduction in cerebral inflammation. There was a significant increase in neuroblasts in the subventricular zone in both treatment groups. The thickness of the most active neurogenic area within the subventricular zone was significantly increased after high-dose treatment compared with CPB and low-dose MSCs, suggesting dose-dependent effects on the neurogenic niche. ConclusionsMSC delivery through CPB is feasible up to 100 × 106 cells per kilogram. MSC treatment during cardiac surgery has the potential to reduce systemic and cerebral inflammation and to modulate responses of an active neurogenic niche to CPB. Further investigation is necessary to assess the long-term effects and to develop a more complete dose-response curve.

The relative biological effectiveness of high-energy clinical 3 and 6 MV X-rays for micronucleus induction in human lymphocytes

Purpose In the modern era of radiotherapy, use of conventional radiation modalities (based on γ-rays) is being replaced by high-energy linear accelerator-based X-rays. As a result of mishandling of equipment or mechanical errors, health workers can be exposed to these high-energy X-rays. Especially in the absence of personnel monitoring devices, biodosimetry with a lower energy X-ray calibration curve may not provide an acceptable dose estimate. Moreover, the relative biological effectiveness (RBE) value assigned for X-rays is the same (ONE) regardless of beam energy (V), employed in diagnosis, interventional medicine, and radiotherapy. Therefore, the purpose of the study is to examine the induced biological effects, measured through micronucleus (MN) formation, of X-rays of different energies (3 and 6 MV X-rays), and to investigate the RBE relative to 225 kVp X-rays. Materials and methods Peripheral blood lymphocytes (PBLs) from healthy donors (n = 6), were irradiated with 225 kVp, 3 MV, and 6 MV energy X-rays and induced biological damage was quantified as MN formation using the cytokinesis blocked MN (CBMN) assay. Results The MN per cell in the X-irradiated samples for the three different X-ray energies showed a significant (p<.0001) dose-dependent increase, when compared to unexposed samples. Aberration frequencies obtained at the same dose for the three different energies showed significant (p<.05) difference for the MN per cell among the energy levels; however, the in vitro dose–response curve parameters (slope, intercept, and coefficient) did not show any significant differences. The estimated dose in the blinded sample was within the 95% confidence intervals of each of the calibration curves. However, overall, the 6 MV dose–response curve coefficients yielded the closest dose estimate to that of the true dose. The calculated RBE values at 5% induced MN for 3 and 6 MV LINAC X-rays were 2.0 ± 0.04 and 0.70 ± 0.01, respectively, and the average RBE for the complete dose–response curves were 1.13 ± 0.04 and 0.80 ± 0.02 relative to 225 kVp X-rays as standard radiation. Conclusion The established dose–response curves obtained for PBL exposed to different energy levels of X-rays of 225 kVp, 3 MV, and 6 MV are ready to use for biological dosimetry purposes. The calculated RBE values for the higher energies of X-rays relative to 225 kVp X-rays in this study suggest that RBE of X-rays may not be equal to one, with the true value dependent on the beam energy, the dose and dose rate, and the endpoint investigated.

A new pharmacodynamic approach to study antibiotic combinations against enterococci in vivo: Application to ampicillin plus ceftriaxone.

The combination of ampicillin (AMP) and ceftriaxone (CRO) is considered synergistic against Enterococcus faecalis based on in vitro tests and the rabbit endocarditis model, however, in vitro assays are limited by the use of fixed antibiotic concentrations and the rabbit model by poor bacterial growth, high variability, and the use of point dose-effect estimations, that may lead to inaccurate assessment of antibiotic combinations and hinder optimal translation. Here, we tested AMP+CRO against two strains of E. faecalis and one of E. faecium in an optimized mouse thigh infection model that yields high bacterial growth and allows to define the complete dose-response relationship. By fitting Hill's sigmoid model and estimating the parameters maximal effect (Emax) and effective dose 50 (ED50), the following interactions were defined: synergism (Emax increase ≥2 log10 CFU/g), antagonism (Emax reduction ≥1 log10 CFU/g) and potentiation (ED50 reduction ≥50% without changes in Emax). AMP monotherapy was effective against the three strains, yielding valid dose-response curves in terms of dose and the index fT>MIC. CRO monotherapy showed no effect. The combination AMP+CRO against E. faecalis led to potentiation (59-81% ED50 reduction) and not synergism (no changes in Emax). Against E. faecium, the combination was indifferent. The optimized mouse infection model allowed to obtain the complete dose-response curve of AMP+CRO and to define its interaction based on pharmacodynamic parameter changes. Integrating these results with the pharmacokinetics will allow to derive the PK/PD index bound to the activity of the combination, essential for proper translation to the clinic.

Ramipril reduces incidence and prolongates latency time of radiation-induced rat myelopathy after photon and carbon ion irradiation.

To test the hypothesis that the use of an angiotensin-converting enzyme inhibitor (ACEi) during radiotherapy may be ameliorative for treatment-related normal tissue damage, a pilot study was conducted with the clinically approved (ACE) inhibitor ramipril on the outcome of radiation-induced myelopathy in the rat cervical spinal cord model. Female Sprague Dawley rats were irradiated with single doses of either carbon ions (LET 45 keV/μm) at the center of a 6 cm spread-out Bragg peak (SOBP) or 6 MeV photons. The rats were randomly distributed into 4 experimental arms: (i) photons; (ii) photons + ramipril; (iii) carbon ions and (iv) carbon ions + ramipril. Ramipril administration (2 mg/kg/day) started directly after irradiation and was maintained during the entire follow-up. Complete dose-response curves were generated for the biological endpoint radiation-induced myelopathy (paresis grade II) within an observation time of 300 days. Administration of ramipril reduced the rate of paralysis at high dose levels for photons and for the first time a similar finding for high-LET particles was demonstrated, which indicates that the effect of ramipril is independent from radiation quality. The reduced rate of myelopathy is accompanied by a general prolongation of latency time for photons and for carbon ions. Although the already clinical approved drug ramipril can be considered as a mitigator of radiation-induced normal tissue toxicity in the central nervous system, further examinations of the underlying pathological mechanisms leading to radiation-induced myelopathy are necessary to increase and sustain its mitigative effectiveness.

Cannabinoid Modulation of Food-Cocaine Choice in Male Rhesus Monkeys.

Marijuana and other cannabinoid compounds are widely used by cocaine users. Preclinical animal studies suggest that these compounds can increase the reinforcing effects of cocaine under some schedules of cocaine self-administration and reinstatement, but not in all cases. To date, no studies have used a food-cocaine concurrent choice procedure, which allows for assessment of drug effects on response allocation, not just changes in cocaine self-administration. The goal of the present study was to examine the effects of compounds differing in their efficacy at the cannabinoid receptor (CBR) on cocaine self-administration using a food-drug choice procedure in monkeys. Four adult male rhesus monkeys were trained to self-administer cocaine in the context of an alternative food (1.0-g banana-flavored pellets) reinforcer, such that complete cocaine dose-response curves (0, 0.003-0.1 mg/kg per injection) were determined each session. Monkeys were tested acutely with the CBR full agonist CP 55,940 (0.001-0.01 mg/kg); the CBR partial agonist Δ9-tetrahydrocannabinol (THC; 0.03-0.3 mg/kg), which is also the primary active ingredient in marijuana and the CBR antagonist rimonabant (0.3-3.0 mg/kg). Cocaine choice increased in a dose-dependent manner. Acute treatment with CP 55,940 decreased cocaine choice, whereas THC and rimonabant enhanced the reinforcing effects of cocaine. Chronic (7-day) treatment with CP 55,940 resulted in tolerance to the decreases in cocaine choice. These findings with Δ9-THC provide support for a potential mechanism for co-abuse of marijuana and cocaine. Additional research with chronic treatment with full CBR agonists on attenuating the reinforcing strength of cocaine is warranted. SIGNIFICANCE STATEMENT: Co-abuse of tetrahydrocannabinol and cocaine is a significant public health problem. The use of animal models allows for the determination of how cannabinoid receptor stimulation or blockade influences the reinforcing strength of cocaine.

Functional random forest with applications in dose-response predictions

Drug sensitivity prediction for individual tumors is a significant challenge in personalized medicine. Current modeling approaches consider prediction of a single metric of the drug response curve such as AUC or IC50. However, the single summary metric of a dose-response curve fails to provide the entire drug sensitivity profile which can be used to design the optimal dose for a patient. In this article, we assess the problem of predicting the complete dose-response curve based on genetic characterizations. We propose an enhancement to the popular ensemble-based Random Forests approach that can directly predict the entire functional profile of a dose-response curve rather than a single summary metric. We design functional regression trees with node costs modified based on dose/response region dependence methodologies and response distribution based approaches. Our results relative to large pharmacological databases such as CCLE and GDSC show a higher accuracy in predicting dose-response curves of the proposed functional framework in contrast to univariate or multivariate Random Forest predicting sensitivities at different dose levels. Furthermore, we also considered the problem of predicting functional responses from functional predictors i.e., estimating the dose-response curves with a model built on dose-dependent expression data. The superior performance of Functional Random Forest using functional data as compared to existing approaches have been shown using the HMS-LINCS dataset. In summary, Functional Random Forest presents an enhanced predictive modeling framework to predict the entire functional response profile considering both static and functional predictors instead of predicting the summary metrics of the response curves.

Evaluation of Anti-cancer activity of Boswellic acid and Montelukast sodium against human prostate cancer cell line PC-3

Prostate cancer is a devastating disease for which current therapies are inadequate. Various lines of evidences have suggested the 5-lipoxygenase (5-LOX) pathway and the leukotriene receptor pathway are potential targets for prevention or treatment of Prostate cancer. Thus, search for new anti-cancer drugs targeting 5-LOX and leukotriene is very essential and important. The objective of the present study was to evaluate the in vitro anti- cancer effects of5-LOX inhibitor-Boswellic acid and cysteinyl leukotrienes receptor antagonist-Montelukast sodium by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay and Sulphorodamine B assay (SRB) assay against human prostate cancer cell line PC-3. Cell viability was assayed by trypan blue dye exclusion assay. A study was carried out in 24, 48 and 72 hrs. In addition, effect of combination of both the above drugs was also determined by same assays. Boswellic acid and Montelukast sodium demonstrated a substantial anti-cancer effect against the PC-3 cell line. The cytotoxicity of both the drugs increased with increase in duration of drug treatment. A combination of both drugs showed significant reduction in cell viability but did not show any synergistic activity. Complete dose-response curves were generated and IC50 values were calculated for all the assays. IC50 Value for Boswellic acid was found to be 49.15- 45.80 µg/ml and 50.14-43.39 µg/ml by MTT assay and SRB assay, respectively at the same time IC50 value for Montelukast sodium was 49.27-46.77 µg/ml and 46.68-46.47 µg/ml by MTT assay and SRB assay respectively. In summary, Boswellic acid and Montelukast sodium are likely to be valuable for the treatment of prostate cancer, but further studies are required for their more extensive biological evaluations.

Differential effects of the dopamine D3 receptor antagonist PG01037 on cocaine and methamphetamine self‐administration in rhesus monkeys

Within the dopaminergic system, the DA D3 receptor (D3R) has been shown to mediate many of the behavioral effects of psychostimulants associated with high abuse potential. The present study sought to extend the assessment of the highly selective D3R antagonist PG01037 on cocaine and methamphetamine (MA) self‐administration to include a food‐drug choice procedure. Complete cocaine and MA self‐administration dose‐response curves were determined daily in each session for eight male rhesus monkeys (n=4/group). When choice was stable, monkeys received acute and five‐day treatment of PG01037 (1.0‐10 mg/kg, i.v.). Acute administration of PG01037 significantly reallocated choice behavior from cocaine to food and decreased cocaine intake, however, tolerance developed by day 5 of treatment. Up to doses that disrupted responding, MA choice and intake were not decreased following acute or 5‐day PG01037 treatment but the behavioral potency was significantly greater on MA choice compared to cocaine choice. Furthermore, using an unconditioned behavior, yawning elicited by the D3/D2R agonist quinpirole (0.003‐0.3 mg/kg) in these same monkeys, the acute efficacy of PG01037 was negatively correlated with the sensitivity to the behavioral effects of quinpirole. These data suggest (1) that efficacy of D3R compounds to decrease drug choice is greater in subjects with lower D3R, perhaps suggesting that it is percent occupancy that is the critical variable in determining efficacy and (2) differences in D3R activity in chronic cocaine vs. MA users. Supported by DA012460, AA007565, and NIDA‐IRP.

A simple procedure for estimating pseudo risk ratios from exposure to non-carcinogenic chemical mixtures

Non-cancer risk assessment traditionally assumes a threshold of effect, below which there is a negligible risk of an adverse effect. The Agency for Toxic Substances and Disease Registry derives health-based guidance values known as Minimal Risk Levels (MRLs) as estimates of the toxicity threshold for non-carcinogens. Although the definition of an MRL, as well as EPA reference dose values (RfD and RfC), is a level that corresponds to "negligible risk," they represent daily exposure doses or concentrations, not risks. We present a new approach to calculate the risk at exposure to specific doses for chemical mixtures, the assumption in this approach is to assign de minimis risk at the MRL. The assigned risk enables the estimation of parameters in an exponential model, providing a complete dose-response curve for each compound from the chosen point of departure to zero. We estimated parameters for 27 chemicals. The value of k, which determines the shape of the dose-response curve, was moderately insensitive to the choice of the risk at the MRL. The approach presented here allows for the calculation of a risk from a single substance or the combined risk from multiple chemical exposures in a community. The methodology is applicable from point of departure data derived from quantal data, such as data from benchmark dose analyses or from data that can be transformed into probabilities, such as lowest-observed-adverse-effect level. The individual risks are used to calculate risk ratios that can facilitate comparison and cost-benefit analyses of environmental contamination control strategies.

Olfactory Responses of Drosophila Larvae

We studied complete dose-response curves for 53 odorants in the third instar larvae of Drosophila melanogaster. All odorants, except one, elicited an attraction response. Some odorants also elicited a decrease from their peak response at higher concentrations. This concentration-dependent decrease in olfactory response could be due to either desensitization or repulsion, 2 possibilities that we cannot distinguish in our current assay. We observed high variations in factors like slopes, thresholds, and peaks of responses that, in agreement with previous studies, suggest that the responses of different receptors are quite different for the similar change in concentration of various ligands. We also observed that lower attraction thresholds predicted higher peak amplitude. This suggests that if odor responses encompassed wider concentration range than can be covered by the dynamic range of a single receptor, then responses tend to be high in magnitude.

Weaker Ligands Can Dominate an Odor Blend due to Syntopic Interactions

Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new “perfume,” or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron’s blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose–response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain.

Determination and Comparison of Graded Dose–Response Curves for Epidural Bupivacaine and Ropivacaine for Analgesia in Laboring Nulliparous Women

The potencies of bupivacaine and ropivacaine have been compared using up-and-down methodology, but their complete dose-response curves have not been compared. The authors performed a random allocation-graded dose-response study of epidural bupivacaine and ropivacaine given epidurally for labor analgesia. Three hundred laboring nulliparous patients were randomly given epidural bupivacaine (5, 10, 15, 20, 30, or 40 mg) or ropivacaine (7, 15, 20, 30, 45, or 60 mg) in 20 ml of saline. Visual Analog Scale pain scores were recorded for 30 min. Response was defined by the percentage decrease in pain score from baseline at 30 min, and dose-response data were analyzed by using nonlinear regression. Sigmoidal Emax model dose-response curves were fitted to the datasets for bupivacaine (R = 0.53) and ropivacaine (R = 0.59). The curves had similar steepness (Hill coefficient 2.02 [95% CI, 1.55-2.50] vs. 2.25 [1.70-2.79], P = 0.55). The ED50 (dose of the drug that reduces pain score to 50% of baseline at 30 min, also known as D50) of ropivacaine was greater than that of bupivacaine (15.3 [95% CI 13.7-17.1] mg vs. 11.3 [10.0-12.7] mg, P = 0.0003), but ED90 (D90) was similar (40.6 [32.4-51.1] mg vs. 33.4 [26.2-42.7] mg, P = 0.29). The potency ratio at ED50 for ropivacaine:bupivacaine was 0.75 (95% CI, 0.65-0.88). Ropivacaine is less potent than bupivacaine, but otherwise they have similar dose-response characteristics. The difference in potency is not statistically significant at ED90 doses.

Measurement of Gastric Acid Secretion in the Anaesthetized Rat

The protocols described in this unit are designed to assess the effects of substances on gastric acid secretion by the rat stomach, with the animal under general anesthesia. Both stimulatory and inhibitory effects of compounds can be evaluated and specific mechanisms of action can also be investigated. Acid secretion is induced by substances that directly activate parietal cell receptors (histamine and bethanechol), by indirect stimuli, like 2-deoxy-D-glucose, by electrical stimulation of vagal nerves, or by the peptide pentagastrin. Reference antisecretory drugs are represented by histamine H(2) receptor antagonists and proton pump inhibitors. This model allows the evaluation of complete dose-response curves together with a time-course of the secretory/antisecretory effects. Indirect effects involving activation or inhibition of vagal pathways are evaluated in intact animals by means of electrical vagal stimulation or in vagotomized animals.

Chlorpyrifos reduces nickel-induced growth retardation of the soil dwelling Collembolan Folsomia candida

For 7 weeks, we studied the effects on body size and growth rate of Folsomia candida exposed to nickel and chlorpyrifos and their mixtures in a natural Lufa 2.2 soil. Nickel significantly reduced the development of body size of the springtails, although no complete dose–response curve was obtained. Chlorpyrifos did not influence the springtail growth when applied alone, but significantly reduced the growth retardation induced by nickel. We showed that by monitoring the development of toxicity in time for both the single compounds and the mixtures it was possible to determine mixture interactions even when no complete dose–response curves were available for all mixture components.

Cytotoxic activity of Brazilian Cerrado plants used in traditional medicine against cancer cell lines

The search for new anti-cancer drugs is one of the most prominent research areas of natural products. Numerous active compounds isolated from Brazilian Cerrado plant species have been studied with promising results. Aim of the study To investigate the cytotoxic potential of 412 extracts from Brazilian Cerrado plants used in traditional medicine belonging to 21 families against tumor cell lines in culture. Material and method Maceration of 50 plant species resulted in 412 hexane, dichloromethane, ethanol and hydroalcohol extracts. The cytotoxicity of the extracts was tested against human colon carcinoma (HCT-8), melanoma (MDA-MB-435), and brain (SF-295) tumor cell lines, using the thiazolyl blue test (MTT) assay. Bioassay-guided fractionation was performed for one active extract. Results and Conclusions Twenty-eight of the 412 tested extracts demonstrated a substantial antiproliferative effect, at least 85% inhibition of cell proliferation at 50 μg/mL against one or more cell lines. Those extracts are obtained from different parts of Anacardiaceae, Annonaceae, Apocynaceae, Clusiaceae, Flacourtiaceae, Sapindaceae, Sapotaceae, Simaroubaceae and Zingiberaceae. Complete dose-response curves were generated and IC 50 values were calculated for these active extracts against four cell lines HCT-8, MDA-MB-435, SF-295 and HL-60 (leukemia), and their direct cytotoxic effects were determined. In summary, 14 extracts of 13 species showed toxicity in all tested tumor cell lines, with IC 50 values ranging from 0.1 to 19.1 μg/mL. The strongest cytotoxic activity was found for the hexane extract of Casearia sylvestris var. lingua stem bark, with an IC 50 of 0.1 μg/mL for HCT-8, 0.9 μg/mL for SF-295, 1.2 μg/mL for MDA-MB-435, and 1.3 μg/mL for HL-60, and Simarouba versicolor root bark, with an IC 50 of 0.5 μg/mL for HCT-8, 0.7 μg/mL for SF-295, 1.5 μg/mL for MDA-MB-435, 1.1 μg/mL for HL-60. Bioassay-guided fractionation of the last extract led to the isolation of glaucarubinone, which showed pronounced activity against the four cell lines studied. Further studies of the active extracts are necessary for chemical characterization of the active compounds and more extensive biological evaluations.

Antiproliferative activity of Hungarian Asteraceae species against human cancer cell lines. Part I

Aqueous and organic extracts of 25 selected species from four tribes of Hungarian Asteraceae were screened in vitro for antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells, using the MTT assay. Twenty five of the 228 tested extracts from different parts of the species of Astereae (6), Inuleae (3), Heliantheae (5) and Anthemideae (11) demonstrated a substantial antiproliferative effect (at least 50% inhibition of cell proliferation) at 10 microg/mL against one or more of the cell lines. Complete dose-response curves were generated and IC(50) values were calculated for these active extracts, and their direct cytotoxic effects were determined. In summary, 11 of the tested 25 plants were found to be active and 4 of them (Anthemis ruthenica, Erigeron canadensis, Erigeron annuus and Inula ensifolia) had not been studied previously for either active compounds or anticancer properties.

Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5

Others have shown that H(2)DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO(3) exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (-160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK (wt, approximately 40 microM) < NKMIK congruent with NKMIN congruent with KKMIN < KNMIN congruent with KNMIK < NNMIK < NNMIN ( approximately 400 microM) < DDMID < EEMIE ( approximately 800 microM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80-91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

Documented optimum and threshold for ionising radiation

A concept of the complete dose-response curve of ionising radiation will allow us to live in harmony with this ubiquitous agent. Convincing data show ionising radiation is essential for life. Ambient levels of ionising radiation (about 2 mSv/y without medical and cosmic radiation) are adequate for life but insufficient for abundant health. We live with a partial deficiency of ionising radiation. Thousands of people have lived for generations with 2?20 times the ambient levels of radiation without showing ill health. A conservative threshold, the maximum safe level of radiation, was estimated from abundant rodent data to be about 8000 mSv/y. When human and rodent data were collated, a conservative optimum of 60 mSv/y was obtained. Radiation levels greater than the threshold are harmful. The facts suggest that radiobiologists and governments should abandon the 'linear no threshold' (LNT) paradigm and accept natural and industrial low level sources of ionising radiation in order to promote abundant health.