Complete Dose-response Relationship Research Articles

Extrapolation of cytotoxic masked effects in planar in vitro assays.

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. ModelledED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose-response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and alsohas applications for other fields of chemical analysis like distorted chromatography signals.

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.

Fish Response to Turbulence Generated Using Multiple Randomly Actuated Synthetic Jet Arrays

Hydroelectric power stations generate turbulent flow conditions, which represent a potentially significant hydraulic stressor to fish passing through the turbine system. A test facility has been developed using two randomly actuated synthetic jet arrays (RASJAs) of 25 independent submersible pumps to generate a turbulent flow field for biological dose-response testing. The novel elements of this approach include the ability to control the exposure duration within a test volume due to low mean flow velocity as well as the capacity to scale the turbulence levels as a function of pump capacity. Juvenile Chinook salmon (Oncorhynchus tshawytscha) were subjected to the turbulent flow regime with average turbulence kinetic energy per unit mass of 0.089 m 2 / s 2 for periods of 2 min and 10 min. No significant loss of equilibrium or disorientation was observed after exposure for either duration at the level of turbulence achieved in this prototype. Further scaling of this approach is required to generate a complete dose-response relationship.

Statistical strategies for averaging EC50 from multiple dose-response experiments.

In most dose-response studies, repeated experiments are conducted to determine the EC50 value for a chemical, requiring averaging EC50 estimates from a series of experiments. Two statistical strategies, the mixed-effect modeling and the meta-analysis approach, can be applied to estimate average behavior of EC50 values over all experiments by considering the variabilities within and among experiments. We investigated these two strategies in two common cases of multiple dose-response experiments in (a) complete and explicit dose-response relationships are observed in all experiments and in (b) only in a subset of experiments. In case (a), the meta-analysis strategy is a simple and robust method to average EC50 estimates. In case (b), all experimental data sets can be first screened using the dose-response screening plot, which allows visualization and comparison of multiple dose-response experimental results. As long as more than three experiments provide information about complete dose-response relationships, the experiments that cover incomplete relationships can be excluded from the meta-analysis strategy of averaging EC50 estimates. If there are only two experiments containing complete dose-response information, the mixed-effects model approach is suggested. We subsequently provided a web application for non-statisticians to implement the proposed meta-analysis strategy of averaging EC50 estimates from multiple dose-response experiments.

Inhibition of Acid Sensing Ion Channel Currents by Lidocaine in Cultured Mouse Cortical Neurons

Lidocaine is a local anesthetic that has multiple pharmacological effects including antiarrhythmia, antinociception, and neuroprotection. Acid sensing ion channels (ASICs) are proton-gated cation channels that belong to the epithelial sodium channel/degenerin superfamily. Activation of ASICs by protons results in sodium and calcium influx. ASICs have been implicated in various physiological processes including learning/memory, nociception, and in acidosis-mediated neuron injury. In this study, we examined the effect of lidocaine on ASICs in cultured mouse cortical neurons. ASIC currents were activated and recorded using a whole-cell patch-clamp technique in cultured mouse cortical neurons. The effects of lidocaine at different concentrations were examined. To determine whether the inhibition of lidocaine on ASIC currents is subunit specific, we examined the effect of lidocaine on homomeric ASIC1a and ASIC2a currents expressed in Chinese hamster ovary cells. Lidocaine significantly inhibits the ASIC currents in mouse cortical neurons. The inhibition was reversible and dose dependent. A detectable effect was noticed at a concentration of 0.3 mM lidocaine. At 30 mM, ASIC current was inhibited by approximately 90%. Analysis of the complete dose-response relationship yielded a half-maximal inhibitory concentration of 11.79 ± 1.74 mM and a Hill coefficient of 2.7 ± 0.5 (n = 10). The effect is rapid and does not depend on pH. In Chinese hamster ovary cells expressing different ASIC subunits, lidocaine inhibits the ASIC1a current without affecting the ASIC2a current. ASIC currents are significantly inhibited by lidocaine. Our finding reveals a new pharmacological effect of lidocaine in neurons.

Passive air sampler as a tool for long-term air pollution monitoring: Part 2. Air genotoxic potency screening assessment

The capability of passive air sampling to be employed in the evaluation of direct genotoxicity of ambient air samples was assessed. Genotoxic effects of the total extracts from the polyurethane foam filters exposed for 28 days during a regional passive air sampling campaign were investigated. Twenty sampling sites were selected in Brno city on the area of approximately 20 × 20 km in October and November 2004. Brno is the second largest city of the Czech Republic, highly industrialized with approximately 370,000 of permanent inhabitants. The levels of PAHs, PCBs, and chlorinated pesticides were determined in all samples. Fraction of each extract was also assayed in the bacterial genotoxicity test using Escherichia coli sulA::lacZ. Complete dose–response relationships of the air extracts were determined. The statistical analysis showed significant correlation between observed biological effects and PAHs concentrations in samples.

Dose-response relationships between herbicides with different modes of action and growth of Lemna paucicostata: an improved ecotoxicological method.

Lemna species are widely used in ecotoxicological research to assess the phytotoxicity of environmental compounds. The purpose of the work described in this paper was to further refine the Lemna spp. bioassay, making it more robust and useful in comparing results between laboratories. A nondestructive image analysis method was used to measure growth as affected by herbicides during a time course. We tested 26 herbicides, with as many as 19 different modes of action, on leaf area growth of Lemna paucicostata to establish complete dose-response relationships. By using 3,5-dichlorophenol as a reference compound, different EC50 (concentration that inhibited growth by 50%) values of the herbicides can be compared. The EC50 values ranged from 0.003 microM for sulcotrione to 407 microM for asulam. Complete dose-response parameters were obtained that will better allow comparison of these results to those of other laboratories. These results should give useful information to anyone who works with Lemna species to address questions on herbicide residues in environmental compartments, screening of new herbicidal compounds, or the assessment of phytotoxic side effects of any compound.

Inhibitory effects of thiol-containing drugs on erythrocyte oxidative damages investigated with an improved assay system

The involvement of oxygen-derived free radicals and other oxidant species in numerous physiopathological processes makes it necessary to develop suitable analytical systems to study their effects and also to assess the antioxidant activity of endogenous and exogenous compounds. In this respect, the properties of three selected thiol-containing drugs (Captopril, N-acetylcysteine and its lysine salt Nacystelyn, a newly developed mucoactive agent) and of reference compounds were examined in a lipid peroxidation model using human red blood cells (RBC) as biological substrate. The thermolabile azo-compound 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH) served for the generation of an oxidative stress and the determination of the extent of RBC haemolysis was recorded. Experimental conditions were developed and optimised to ensure the stability and reproducibility of the system and to establish complete dose–response relationships in order to determine relevant pharmacological parameters. Actually, the AAPH/haemolysis system shrewdly combined with a procedure to measure the extent of haemolysis in which all common haemoglobin derivatives released following haemolysis are converted to cyanomethaemoglobin, allowed the assessment of the antioxidant activity of most investigated drugs. Precision was also improved by considering readings at 50% haemolysis ( T 50). The following sequence was obtained for the antioxidant properties of investigated drugs: uric acid>Trolox>ascorbic acid> N-acetylcysteine∼Nacystelyn>Captopril≫ l-lysine.

Characterization of dioxin‐like activity of sediments from a Czech River Basin

Synthetic organic chemicals are present in environmental compartments as complex mixtures and therefore their potential effects are difficult to predict. In this study, in vitro bioassays using wild-type fish and rat hepatoma cell lines and their corresponding recombinant cell systems were used to evaluate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity in extracts of sediments collected from rivers of the Czech Republic. All the sediment extracts elicited statistically significant responses in all the cell lines tested. For most sediment extracts, a complete dose-response relationship was obtained. The maximal efficacy of the samples was between 57 and 143% of the maximal induction elicited by TCDD. Greater responsiveness, sensitivity, and reproducibility were observed for recombinant than wild-type cells. Cell line-specific differences in the sensitivity to compounds present in the complex sediment extracts were observed. The TCDD equivalents (TCDD-EQs) determined from the different cell bioassays were correlated. Greater concentrations of TCDD-EQs were obtained with fish cell lines. The TCDD-EQs calculated from the results of chemical analysis of toxic equivalents (TEQs) were in good agreement with those determined by bioassays; the arly hydrocaron receptor (AhR)-effects of the identified chemicals appear to be generally additive. This indicates that most of the TCDD-like activity was accounted for by the compounds identified and quantified by instrumental analysis. Fractionation along with mass-balance calculations allowed identification of the active fractions and classes of compounds. Polycyclic aromatic hydrocarbons (PAHs) were found to be responsible for most of the AhR-mediated activity in sediments.

Quantitative carcinogenesis and dosimetry in rainbow trout for aflatoxin B 1 and aflatoxicol, two aflatoxins that form the same DNA adduct

Two exposure protocols were used to establish complete dose-response relationships for the hepatic carcinogenicity and DNA adduction in vivo of aflatoxin B 1 (AFB 1) and aflatoxicol (AFL) in rainbow trout. By passive egg exposure, AFL was taken up less well than AFB 1, but was more efficiently sequestered into the embryo itself, to produce an embryonic DNA bibding curve that was linear with carcinogen dose and with a DNA binding index three-fold greater than AFB 1. Both aflatoxins produced the same phenotypic response, predominantly mixed hepatocellular/cholangiocellular carcinoma. Tumor responses as logit [incidince] vs. In [dose] were parallel-offset, non-linear responses showing a three-fold greater carcinogenic potency for AFL at all doses examined (i.e. 3 times more AFB 1 than AFL required to produce an equivalent liver tumor incidence). By molecular dosimetry analysis (logit [incidence] vs. In [DNA adducts]), the two data sets were coincident, indicating that, per DNA adduct formed in vivo in total embryonic DNA, these two aflatoxins were equally efficient in tumor initiation. By dietary fry exposure, both carcinogens produced linear DNA binding dose responses in liver, but with an AFL target organ DNA binding index only 1.14 times that of AFB 1 by this exposure route. The tumor dose-response curves also did not exhibit the three-fold difference shown by embryo exposure, but were closely positioned non-linear curves. Since the DNA binding indices differed by only 14%, the resulting molecular dosimetry curves for AFL and AFB 1 by dietary exposure were similar to the tumor response curves. These results indicate that differing exposure routes produced differing relative carcinogenicity estimates based on doses appllied result of protocol-dependent differences in AFL and AFB 1 pharmacokinetic behaviors, but that potency comparisons based on molecular dose received were similar for the two protocols. By comparison with standard DNA adducts produced in vitro using the dimethyloxirane-produced 8,9-epoxides of AFB 1 and AFL, we conclude that > 99% of AFL-DNA adducts produced in vivo were identical to those produced by AFB 1. Thus similar molecular dosimetry responses should be expected under all exposure protocols in which the two parent carcinogens do not exhibit differing toxicities to the target organ.

Tumor and normal tissue response to interstitial photodynamic therapy of the rat r-1 rhabdomyosarcoma

Interstitial photodynamic therapy (IPDT) using Photofrin II (PII) as photosensitizer has been studied in the rat rhabdomyosarcoma R-1, growing on the thigh or flank of WAG-Rij rats. A light dose-response relationship has been established, for 10 mg PII/kg i.v. and irradiation 24 hr later, with local tumor control as the end point for single IPDT treatments using four cylindrical diffusors simultaneously. A light energy fluence of 150–200 Joule/ cm 2 (wavelength 625 nm), measured in vivo at the tumor periphery, was required for tumor control. Comparison of tumor response at 5 and 2.5 mg PII/kg with the complete dose response relationship at 10 mg PII/kg suggests drug-light dose reciprocity and indicates that in our tumor model treatment failures are not likely to be caused by variations in (tumor) tissue photosensitizer level, but rather by insufficient light dose or inadequate light dose distribution. Increasing the interval between PII administration and irradiation from 24 hr to 48 hr had no great effect on tumor response to IPDT in this study. Inspection of the original tumor site 100 days after tumor', control revealed obvious loss of thigh muscle tissue. Also, recurrent tumors showed a reduced growth rate. Therefore, the relationship between tumor (re)growth and PDT-induced normal tissue damage was studied and the existence of a tumor bed effect was confirmed. The present study indicates that tumor control after a single IPDT treatment is feasible, but that PDT induced damage to a margin of the adjacent normal tissue is probably required.

Cardiovascular effects of L-tyrosine in normotensive and hypertensive rats

There are conflicting reports on the blood pressure effects of tyrosine. The aim of this study was to establish complete dose-response relationships and to compare the effects of various modes of administration of L-tyrosine in anaesthetised normotensive and spontaneously hypertensive rats. The intravenous injection of L-tyrosine, 0.2–0.4 mmol/kg, produced tachycardic and hypertensive effects in both species. The higher doses (0.8–1.6 mmol/kg) produced marked bradycardiac and hypotensive responses. Intracerebroventricular administration of L-tyrosine, 0.005–0.1 mmol/kg, had no statistically significant effects. Chronic dietary administration of L-tyrosine, approximate daily doses of 0.7–55 mmol/kg was also without any significant effects. These results suggest that the controversies in the earlier studies could be due mainly to differences in doses and modes of administration. Our results also suggest that the cardiovascular effects of tyrosine are peripheral rather than central in origin although a central site of action cannot be excluded.

Internal and external carotid vascular responses to vasoactive agents in the monkey

Internal and external carotid blood flows in anesthetized monkeys were measured simultaneously using electromagnetic flowmeters. Complete dose-response relationships were established for the effects of intracarotid infusion of several humoral agents implicated in migraine. Both the internal and external carotid vasculatures were constricted by serotonin and prostaglandin F2alpha and dilated by bradykinin, histamine, and acetylcholine. Noradrenalin and adrenaline constricted the external carotid vasculature but had little direct effect in the internal carotid territory. Prostaglandin E1 dilated the external carotid vasculature. Low doses of prostaglandin E1 produced dilation in the internal carotid circulation, but with higher doses there was a paradoxical abolition of this effect.

Behavioral effects of LSD in the cat: Proposal of an animal behavior model for studying the actions of hallucinogenic drugs

In the course of examining the complete dose-response relationship for the behavioral effects of LSD in the cat, we discovered that, in addition to large increases in investigatory and hallucinatory-like responses, two behaviors, not previously reported, are emitted with a high probability under LSD. Beginning from a baseline of essentially zero in saline-treated animals, limb flicks and abortive grooming increase in frequency in direct relation to the dose of LSD administered (2.5, 10, 25 and 50 μg/kg i.p.) and then decrease at higher doses (100 and 200 μg/kg). Limb flicks are a species-specific behavior seen in normal cats almost exclusively in response to the presence of a foreign substance, such as water, on the hindpaw or forepaw. In abortive grooming, the cat orients to the body surface as if to groom but does not emit the consummatory grooming response (bite, lick or scratch), or emits the response in midair. These behaviors can serve as an animal behavior model for the actions of LSD and related hallucinogens in humans. The specificity of these behavioral changes is indicated by the fact that they are never seen in response to other classes of psychoactive drugs such as d-amphetamine, atropine, caffeine and chlorpheniramine. They are, however, elicited by compounds such as psilocybin which are structurally and functionally related to LSD. The validity of the model is based on evidence indicating that it is: specific to hallucinogens, dose dependent, observed in a dose range effective in humans, parallels the major parameters of the actions of LSD in humans (see following paper), sensitive, robust, reliable, quantifiable and easy to score.

Selective microvascular constrictor actions of some neurohypophyseal peptides

The present experiments, using an image-splitting television microscope recording system, show for the first time that it is possible to obtain quantitative, complete dose—response relationships for the constrictor actions of a series of vasopressin and oxytocin hormone analogues on all major types of microscopic blood vessels, in situ, in the terminal vascular bed. The findings demonstrate that: (1) the structure—action relationships for neurohypophyseal peptide-induced contractions on microscopic blood vessels may vary with the particular type of muscular microvessel (i.e., arteriole, venule, metarteriole, precapillary sphincter); (2) although basicity in position 8 of the vasopressin hormones is probably important for hormone-receptor affinity on all mammalian somatic blood vessels, including those in the microcirculation, it is not important for intrinsic (contractile) activity; and (3) certain synthetic analogues of the neurohypophyseal peptide hormones exert selective microvascular constrictor actions. Thus, the present data show that some previously synthesized neurohypophyseal analogues exhibit relatively high hormone-receptor affinities (H-RA) for particular types of microvessels while exhibiting very low H-RA for other types of microscopic blood vessels. It is suggested that a heterogeneity of the vasopressin receptor, in mammalian vascular smooth muscle cells, of different types of blood vessels may exist.

Fast Neutron Irradiation and the Relationship of Radiation Dose and Mammalian Tumor Cell Reproductive Capacity

The dose-response relationship for neutron irradiation of a quantitative in vivo mouse tumor cell system was determined and compared with the dose- response relationship for x irradiation performed under both anoxic and oxygenated conditions. Values of n = l.0 and D/sub 37/. = 85 rads were obtained for fission-spectrum fast neutron irradiation as compared with n = 1.6 and D/sub 37/ = 365 rads for x irradiation under anoxic conditions and n = l.6 and D/su b 37/ = I60 rads for x irradiation under oxygenated conditions. The cell system employed conforms in its radiobiological behavior with other mammalian cell culture systems and constitutes a useful test system for the study of cell radiobiological phenomena. The same or greater degree of apparent radiosensitivity was attained by direct neutron irradiation of cells in the anoxic state as was attained by x irradiation in the oxygenated state. Radiosensitivity cannot be defined in terms of a single RBE ratio but only in terms of the complete dose-response relationship or in respect to the parameters, n and D/sub 37/, which describe this relationship, (auth)