Non-parallel Dose-response Curves Research Articles

A Genomics-Based Analysis of Relative Potencies of Dioxin-Like Compounds in Primary Rat Hepatocytes

Toxic equivalency factors (TEFs) for dioxin-like compounds are largely based on relative potency (REP) values derived from biochemical endpoints such as enzyme activity. As of yet, REPs based on gene expression changes have not been accounted for in the TEF values. In this study, primary rat hepatocytes were treated for 24h with 11 concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) ranging from 0.00001 to 100 nM. Differential changes in gene expression were analyzed using analysis of variance to assess the relative contributions of concentration, congener, and the interaction between concentration and congener for each gene. A total of 3283 genes showed significant changes with concentration (false discovery rate < .05 and fold-change ± 1.5 in at least 1 concentration for 1 congener). Among these genes, 399 were significant for both concentration and congener effects indicating parallel concentration-response curves with significant differences in potency. Only 8 genes showed a significant concentration and congener interaction term indicating a minority of genes show nonparallel dose-response curves among the 3 congeners. Benchmark dose (BMD) modeling was used to derive BMD values for induced individual genes and signaling pathways. The REP values for 4-PeCDF and TCDF were generally 3- to 5-fold lower than the World Health Organization (WHO) TEF values on both a gene and pathway basis. These findings suggest that the WHO TEF values may possibly overpredict the potency of these polychlorinated dibenzofuran congeners and demonstrate the importance of identifying functional pathways relevant to the toxicological modes of action for establishing pertinent REPs.

Toxicodynamic analysis of the combined cholinesterase inhibition by paraoxon and methamidophos in human whole blood

Theoretical work has shown that the isobole method is not generally valid as a method for testing the absence or presence of interaction (in the biochemical sense) between chemicals. The present study illustrates how interaction can be tested by fitting a toxicodynamic model to the results of a mixture experiment. The inhibition of cholinesterases (ChE) in human whole blood by various dose combinations of paraoxon and methamidophos was measured in vitro. A toxicodynamic model describing the processes related to both OPs in inhibiting AChE activity was developed, and fit to the observed activities. This model, not containing any interaction between the two OPs, described the results from the mixture experiment well, and it was concluded that the OPs did not interact in the whole blood samples. While this approach of toxicodynamic modeling is the most appropriate method for predicting combined effects, it is not rapidly applicable. Therefore, we illustrate how toxicodynamic modeling can be used to explore under which conditions dose addition would give an acceptable approximation of the combined effects from various chemicals. In the specific case of paraoxon and methamidophos in whole blood samples, it was found that dose addition gave a reasonably accurate prediction of the combined effects, despite considerable difference in some of their rate constants, and mildly non-parallel dose–response curves. Other possibilities of validating dose-addition using toxicodynamic modeling are briefly discussed.

Using translational medicine to understand clinical differences between botulinum toxin formulations

When using botulinum toxin-based products, the physician must decide the optimal location and dose required to alleviate symptoms and improve the patient's quality of life. To deliver effective treatment, the physician needs to understand the importance of accurate target muscle selection and localization and the implications of each product's migration properties when diluted in different volumes. Pre-clinical mouse models of efficacy and safety have been utilized to compare local and distal muscle relaxation effects following defined intramuscular administration. Data from the model allow the products to be ranked based on their propensity for local efficacy versus their distal migration properties. Using standardized dilutions, the non-parallel dose-response curves for the various formulations demonstrate that they have different efficacy profiles. Distal effects were also noted at different treatment doses, which are reflected in the different safety and/or therapeutic margins. Based on these pre-clinical data, the safety and therapeutic margin rankings are ordered, largest to smallest, as BOTOX, Dysport and Myobloc. The results of subsequent clinical trials are variable and dose comparisons are inconclusive, thus supporting the regulatory position that the dose units of the individual preparations are unique and cannot be simply converted between products.

Analytical performance of serum free light-chain assay during monitoring of patients with monoclonal light-chain diseases

BackgroundMeasurement of serum free light chains (FLC) is useful for the diagnosis and monitoring of monoclonal light-chain diseases. It has been suggested that there will be widespread replacement of urine Bence Jones protein measurement by serum FLC assay. We report on our experience with the assay during monitoring of light-chain myeloma (LCMM) and AL amyloidosis (AL). MethodsSerum FLC immunoassay, serum and/or urine protein electrophoresis and immunofixation were performed on serial samples during monitoring of LCMM. Recovery and immunoreactivity of FLC were tested by sample dilution. Assay precision was determined by repeat assay of samples over several reagent lots. ResultsIn one of 23 patients with LCMM there was non-reaction of a monoclonal kappa FLC with some reagent lots and the assay did not indicate disease relapse. Samples showed non-linear, non-parallel immunoreactivity on dilution. Several tested monoclonal FLC gave lower values at the assay starting dilution compared with higher sample dilution and non-parallel dose–response curves. The median between-reagent lot variation for FLC measurement was 19–20% CV. ConclusionsLaboratory staff and clinicians need to be aware of the potential for non-reactivity of individual monoclonal FLC, and the effects of dilution and precision on FLC values and their interpretation.

Quantitation of IgG subclass antibody responses after immunization with a group B meningococcal outer membrane vesicle vaccine, using monoclonal mouse-human chimeric antibodies as standards

An ELISA method was developed to quantitate gravimetrically (μg/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1 3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

Relative potency of polychlorinated dibenzo‐p‐dioxins and dibenzofurans for inducing mixed‐function oxygenase activity in rainbow trout

AbstractToxic equivalent factors (TEFs) were generated for various polychlorinated dibenzo‐p‐dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) to compare the relative potencies as inducers of liver mixed‐function oxygenase (MFO) activity in rainbow trout (Oncorhynchus mykiss). Trout, dosed orally with one of five PCDD congeners, either [3H]‐2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), l,2,3,7,8‐pentachlorodibenzo‐p‐dioxin (PnCDD), 1,2,3,6,7,8‐hexachlorodibenzo‐p‐dioxin (1,2,3,6,7,8‐HxCDD), or [14C]‐1,2,3,4,7,8‐hexachlorodibenzo‐p‐dioxin (1,2,3,4,7,8‐HxCDD), or [14C]‐1,2,3,4,6,7,8‐heptachlorodibenzo‐p‐dioxin (HpCDD) or one of four PCDF congeners, either 2,3,7,8‐tetrachlorodibenzofuran (TCDF), 1,2,3,7,8‐pentachlo‐rodibenzofuran (1,2,3,7,8‐PnCDF), 2,3,4,7,8‐pentachlorodibenzo furan (2,3,4,7,8‐PnCDF), or 1,2,3,4,7,8‐hexachlorodibenzofuran (HxCDF), showed increases in liver ethoxyresorufin O‐deethylase (EROD) activity up to 250‐fold. Liver concentrations of congeners were better predictors of EROD activity than oral dose. TEFs, ratios of the potency of each congener to that of TCDD, were calculated comparing “threshold” PCDD concentrations that induced EROD significantly above control levels. In cases of nonparallel dose‐response curves between congeners, these comparisons are more relevant to environmental situations as PCDDs are compared at concentrations closer to those in the environment, instead of at higher pharmaceutical levels. Threshold‐based TEFs were: PnCDD, 1.8; 1,2,3,6,7,8‐HxCDD and 1,2,3,4,7,8‐HxCDD, 0.4; HpCDD, 0.05; 2,3,4,7,8‐PnCDF, 2; TCDF, 0.5; 1,2,3,7,8‐PnCDF and HxCDF, 0.4. The ranking of potencies of the PCDFs was the same in fish as in mammalian tests, with the exception of PnCDD and 2,3,4,7,8‐PnCDF, which were more potent to fish than was TCDD. Fish TEFs calculated at the threshold of EROD induction were about four to five times larger than international TEFs (I‐TEFs), which are based on mammalian tests. At low concentrations typical of environmental exposures, TEFs may be different from mammalian‐based TEFs.

Laboratory Selection of Haemonchus contortus for Resistance to Ivermectin

The eighth generation of adult Haemonchus contortus, selected by subjecting infected pairs of sheep to suboptimal ivermectin treatment once per generation from parent (P; BBH isolate) through F7 (IV-A; selected isolate), required an approximate 4-fold increase in the ivermectin dose to produce 95% efficacy compared with its contemporary parent isolate. In a dose titration experiment the dose-response curve of the drug pressure-derived isolate, IV-A, was significantly (0.02 less than P less than 0.05) less steep than was the response curve of the parent, BBH, isolate. Potency estimates based upon these nonparallel dose-response curves would not remain constant over a range of efficacy levels but would decrease rapidly at efficacies greater than 95%. Passage of a closed population of the F8 generation of IV-A sequentially through pairs of sheep for an additional 11 generations (F8A-F8K) without additional drug pressure being applied produced no reversion to sensitivity to ivermectin relative to the F7 generation, thus suggesting that the selected "resistance" was stable.

Cardiovascular effects of bevantolol, a selective beta 1-adrenoceptor antagonist with a novel pharmacological profile.

Bevantolol was more potent in blocking the chronotropic than the hypotensive effects of isoprenaline in pithed rats. Bevantolol itself induced bradycardia, so that it was not possible to estimate the pA2 from nonparallel dose-response curves relating isoprenaline concentration to tachycardia. Bevantolol caused hypertension in pithed rats, an effect attenuated by phentolamine, implying that bevantolol may be an alpha-adrenoceptor agonist. Bevantolol potentiated the pressor effects of noradrenaline, the maximum potentiation equalling that produced by prior chemical sympathectomy with guanethidine, implying that bevantolol may block noradrenaline uptake. In isolated atria bevantolol-induced bradycardia was associated with a positive shift in take-off potential, a reduction in the maximum rate of depolarization (Vmax), and a lengthening of action potential duration (APD). No change in the slope of the slow diastolic depolarization occurred except at the highest concentration (18 mumol l(-1). In atrial and ventricular muscle bevantolol reduced Vmax and overshoot potential, implying reduction of fast inward sodium current (Class I antiarrhythmic action). In pithed rats bevantolol lengthened the P-R interval in the ECG, and produced atrioventricular (A-V) block, and bundle-branch block. In isolated A-V nodal preparations, intranodal conduction time was greatly increased, implying restriction of inward current through calcium channels responsible for nodal depolarization. Bevantolol had no negative inotropic effect in pithed rats, or in isolated atria, and did not alter the positive inotropic effect of raised extracellular calcium concentration, implying absence of restriction of current through calcium channels controlling contraction of the myocardium.

Lack of correlation between factor VIII related antigen multimeric analysis pattern and parallel or non-parallel dose response curves in an ELISA factor VIII related antigen assay.

Factor VIII related antigen has been measured and epitope distribution has been explored by testing the degree of parallelism between standard and test plasma dose response curves using an enzyme immunoassay. Normal plasma, plasma fractions, and plasma from patients with haemophilia and von Willebrand's disease have been tested. All showed parallelism except for plasma from patients with the variant type IIA von Willebrand's disease, of which 10 had parallel and five had non-parallel dose response curves when compared with that of normal plasma. In one family plasma from seven members showed parallelism but from four others did not. An unrelated patient was tested on three occasions, and although the samples were parallel to each other, no sample was parallel to the standard. No correlation was found between parallelism as shown by the enzyme immunoassay and differences in factor VIII related antigen multimeric pattern, including triplet configuration, seen in the type IIA patients.

BIOLOGICALLY ACTIVE LUTEINIZING HORMONE (LH) IN PLASMA

ABSTRACT A recently described in vitro bioassay method for the measurement of biologically active LH (Van Damme et al. 1974) has been applied to the plasma of normally menstruating and post-menopausal women. The specificity of the procedure was established according to the following evidence: 1. Parallelism was observed between dose response curves obtained with serial dilutions of a standard LH preparation (HMG 2nd IRP) and plasma pools collected during the follicular phase, at the LH-peak, during the luteal phase and after menopause. 2. There was no evidence for the presence of any synergistic or antagonistic factor in the various plasma specimens. The assay design used to establish this consisted of assaying the standard and plasma pool separately and then together as a mixture followed by an asssessment of the difference (if any) in the potencies obtained. Strict additivity should yield a relative potency of 1.0. Plasma pools which were obtained every 2–3 days throughout the menstrual cycle were assayed using this design against the standard (HMG 2nd IRP) and against a mid-cycle plasma pool obtained at the time of the LH-peak. The latter was also assayed against partially purified plasma fractions obtained from a post-menopausal plasma pool after gel filtration and isoelectric focusing. With the exception of 3 assays, in which the estimates of relative potency were 0.91, 0.94 and 0.95, respectively, in 19 assays of additivity, the fiducial limits always included unity. 3. Non-detectable LH levels were found in the plasma or serum of either hypophysectomized or hypopituitary hypogonadal men or women treated with oestrogen/progestogen combined pills. 4. The presence of calf or human serum in the assay medium is an essential requirement for a valid comparison of standard and unknown preparations. In their absence, non-parallel dose response curves between plasma and standard were obtained. The other established criteria of reliability (sensitivity and precision) were also examined. The method is sufficiently sensitive (3.5–8.0 mIU/ml plasma; HMG (2nd IRP) as standard) for the measurement of LH throughout the cycle. The mean index of precision (λ) in 230 multiple assays was 0.040. It is concluded that the modified bioassay yields valid and reliable estimates of LH when applied to human plasma obtained throughout the menstrual cycle and after menopause.

A comparison of the effects of angiotensin II and heptapeptide on smooth muscle (vascular and uterine)

On the rabbit isolated aorta, dose-dependent contractions to both angiotensin II and heptapeptide ([des-Asp 1]-angiotensin II) were obtained. The curves were parallel, and reached the same maximum level. On the rat isolated uterus, angiotensin II and the heptapeptide displayed non-parallel dose—response curves. Results obtained with angiotensin-analog antagonists and cross-tachyphylaxis experiments suggest that the heptapeptide and angiotensin II act, preferentially, on different populations of receptors in the uterus. The difference in action of indomethacin on recovery from tachyphylaxis to angiotensin II and heptapeptide on rat isolated aorta suggests that the mechanism of induction of tachyphylaxis by these two peptides may differ. SQ 20881, the angiotensin converting enzyme inhibitor, totally inhibites uterine responses to both decapeptide and nonapeptide, while slightly potentiating those to angiotensin II and heptapeptide. Indomethacin had no significant effect on uterine responses to either angiotensin II or the heptapeptide.