EC50 Ratio Research Articles

High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine.

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 μM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Design and Synthesis of Orexin 1 Receptor-Selective Agonists.

Orexins are a family of neuropeptides that regulate various physiological events, such as sleep/wakefulness as well as emotional and feeding behavior, and that act on two G-protein-coupled receptors, i.e., orexin 1 (OX1R) and orexin 2 receptors (OX2R). Since the discovery that dysfunction of the orexin/OX2R system causes the sleep disorder narcolepsy, several OX2R-selective and OX1/2R dual agonists have been disclosed. However, an OX1R-selective agonist has not yet been reported, despite the importance of the biological function of OX1R. Herein, we report the discovery of a potent OX1R-selective agonist, (R,E)-3-(4-methoxy-3-(N-(8-(2-(3-methoxyphenyl)-N-methylacetamido)-5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)-N-(pyridin-4-yl)acrylamide [(R)-YNT-3708; EC50 = 7.48 nM for OX1R; OX2R/OX1R EC50 ratio = 22.5]. The OX1R-selective agonist (R)-YNT-3708 exhibited antinociceptive and reinforcing effects through the activation of OX1R in mice.

Discovery of AL-GDa62 as a Potential Synthetic Lethal Lead for the Treatment of Gastric Cancer.

Diffuse gastric cancer and lobular breast cancer are aggressive malignancies that are frequently associated with inactivating mutations in the tumor suppressor gene CDH1. Synthetic lethal (SL) vulnerabilities arising from CDH1 dysfunction represent attractive targets for drug development. Recently, SLEC-11 (1) emerged as a SL lead in E-cadherin-deficient cells. Here, we describe our efforts to optimize 1. Overall, 63 analogues were synthesized and tested for their SL activity toward isogenic mammary epithelial CDH1-deficient cells (MCF10A-CDH1-/-). Among the 26 compounds with greater cytotoxicity, AL-GDa62 (3) was four-times more potent and more selective than 1 with an EC50 ratio of 1.6. Furthermore, 3 preferentially induced apoptosis in CDH1-/- cells, and Cdh1-/- mammary and gastric organoids were significantly more sensitive to 3 at low micromolar concentrations. Thermal proteome profiling of treated MCF10A-CDH1-/- cell protein lysates revealed that 3 specifically inhibits TCOF1, ARPC5, and UBC9. In vitro, 3 inhibited SUMOylation at low micromolar concentrations.

Potential modulating effect of the Ascaris suum nicotinic acetylcholine receptor (nAChR) by compounds GSK575594A, diazepam and flumazenil discovered by structure-based virtual screening approach

Parasitic infections are a widespread health problem and research of novel anthelmintic compounds is of the utmost importance. In this study we performed a virtual screening campaign by coupling ligand-based pharmacophore, homology modeling and molecular docking. The virtual screening campaign was conducted using a joined pool of the Drugbank database and a library of purchasable compounds in order to identify drug like compounds with similar pharmacological activity. Our aim was to identify compounds with a potential antihelmintic modulatory effect on nicotinic acetylcholine receptors (nAChR). We derived a 3D pharmacophore model based on the chemical features of known Ascaris suum nAChR modulators. To evaluate the in silico predictions, we tested selected hit compounds in contraction assays using somatic muscle flaps of the Ascaris suum neuromuscular tissue. We tested the modulatory effects of GSK575594A, diazepam and flumazenil hit compounds on nematode contractions induced by acetyl choline (ACh). The compound GSK575594A (3 μM) increased the Emax by 21 % with the EC50 dose ratio of 0.96. Diazepam (100 μM) decreased the Emax by 15 % (1.11 g to 0.95 g) with the EC50 ratio of 1.42 (shifted to the left from 11.25 to 7.93). Flumazenil decreased the EC50 value (from 11.22 μM to 4.88 μM) value showing dose ratio of 2.30, and increased the Emax by 4 % (from 1.54 g to 1.59 g). The observed biological activity was rationalized by molecular docking calculations. Docking scores were calculated against several binding sites within the Ascaris suum homology model. We constructed the homology model using the ACR-16 subunit sequence. The compound GSK575594A showed strong affinity for the intersubunit allosteric binding site within the nAChR transmembrane domain. The binding modes of diazepam and flumazenil suggest that these compounds have a comparable affinity for orthosteric and allosteric nAChR binding sites. The selected hit compounds displayed potential for further optimization as lead compounds. Therefore, such compounds may be useful in neutralizing the growing resistance of parasites to drugs, either alone or in combination with existing conventional anthelmintics.

Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system

Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5–10 % depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50 % maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13–2.01 μM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.

Carvacrol acts as a potent selective antagonist of different types of nicotinic acetylcholine receptors and enhances the effect of monepantel in the parasitic nematode Ascaris suum

The neuromuscular system of parasitic nematodes has proven to be an efficient pharmacological target for antihelmintics. Some of the most frequently used antiparasitic drugs are agonists or antagonists of nicotinic acetylcholine receptors (nAChRs). The antinematodal mechanism of action of carvacrol involves the inhibition of parasite muscle contraction. We have examined the interaction of carvacrol with antinematodal drugs that are agonists of different subtypes of nAChRs and monepantel, which is a non-competitive antagonist of this receptor in A. suum. Additionally, we investigated the effect of carvacrol on the muscle type of nAChRs in the mammalian host. As orthosteric agonists of nAChR, pyrantel, morantel and befinijum lead to dose-dependent contractions of the neuromuscular preparation of Ascaris suum. Carvacrol 100 μM decreased the Emax of pyrantel, morantel and bephenium by 29%, 39% and 12 %, 39 % and 12 % respectively. The EC50 ratio was 3.43, 2.95 and 2.47 for pyrantel, morantel and bephinium, respectively. Carvacrol 300 u μM reduces the Emax of pyrantel, morantel and bephenium by 71%, 80% and 75 %, 80 % and 75 % respectively. The EC50 ratio for pyrantel, morantel and bephenium was 3.88, 3.19 and 4.83 respectively. Furthermore, carvacrol enhances the inhibitory effect of monepantel on A. suum contractions, which may have an effective clinical application. On the other hand, tested concentrations of carvacrol did not significantly affect the EFS-induced contractions of the rat diaphragm, indicating a lack of interaction with the postsynaptic nAChR at the muscle end plate in mammals, but the highest concentration (300 μM) caused a clear tetanic fade. Carvacrol exhibited a time and dose-dependent effect on the Rota-rod performances of rats with a high value of the ED50 (421.6 mg/kg). In our research, carvacrol dominantly exhibited characteristics of a non-competitive antagonist of nAChR in A. suum, and enhances the inhibitory effect of monepantel. The combination of monepantel and carvacrol may be clinically very effective, and the carvacrol molecule itself can be used as a promising platform for the development of new anthelmintic drugs.

Investigation of the Synergistic Toxicity of Binary Mixtures of Pesticides and Pharmaceuticals on Aliivibrio fischeri in Major River Basins in South Korea.

This work introduces the potential synergistic toxicity of binary mixtures of pesticides and pharmaceuticals, which have been detected in substantial amounts in major river basins in South Korea. Different dose-response curve functions were employed in each experimental toxicity dataset for Aliivibrio fischeri. We tested the toxicity of 30 binary mixtures at two effect concentrations: high effect concentration [EC50] and low effect concentration (EC10) ranges. Thus, the toxicological interactions were evaluated at 60 effected concentration data points in total and based on model deviation ratios (MDRs) between predicted and observed toxicity values (e.g., three types of combined effects: synergistic (MDR > 2), additive (0.5 ≤ MDR ≤ 2), and antagonistic (MDR < 0.5)). From the 60 data points, MDRs could not be applied to 17 points, since their toxicities could not be measured. The result showed 48%-additive (n = 20), 40%-antagonistic (n = 17), and 12%-synergistic (n = 6) toxicity effects from 43 binaries (excluding the 17 combinations without MDRs). In this study, EC10 ratio mixtures at a low overall effect range showed a general tendency to have more synergistic effects than the EC50 ratio mixtures at a high effect range. We also found an inversion phenomenon, which detected three binaries of the combination of synergism at low concentrations and additive antagonism at high concentrations.

Affinity, potency, efficacy, and selectivity of neurokinin A analogs at human recombinant NK2 and NK1 receptors

A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [125I]-NKA binding to NK2 receptors and displacement of [3H]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [β-Ala8]-NKA(4–10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys5,MeLeu9,Nle10]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8).

Global concentration additivity and prediction of mixture toxicities, taking nitrobenzene derivatives as an example

The toxicity of a mixture depends not only on the mixture concentration level but also on the mixture ratio. For a multiple-component mixture (MCM) system with a definite chemical composition, the mixture toxicity can be predicted only if the global concentration additivity (GCA) is validated. The so-called GCA means that the toxicity of any mixture in the MCM system is the concentration additive, regardless of what its mixture ratio and concentration level. However, many mixture toxicity reports have usually employed one mixture ratio (such as the EC50 ratio), the equivalent effect concentration ratio (EECR) design, to specify several mixtures. EECR mixtures cannot simulate the concentration diversity and mixture ratio diversity of mixtures in the real environment, and it is impossible to validate the GCA. Therefore, in this paper, the uniform design ray (UD-Ray) was used to select nine mixture ratios (rays) in the mixture system of five nitrobenzene derivatives (NBDs). The representative UD-Ray mixtures can effectively and rationally describe the diversity in the NBD mixture system. The toxicities of the mixtures to Vibrio qinghaiensis sp.-Q67 were determined by the microplate toxicity analysis (MTA). For each UD-Ray mixture, the concentration addition (CA) model was used to validate whether the mixture toxicity is additive. All of the UD-Ray mixtures of five NBDs are global concentration additive. Afterwards, the CA is employed to predict the toxicities of the external mixtures from three EECR mixture rays with the NOEC, EC30, and EC70 ratios. The predictive toxicities are in good agreement with the experimental toxicities, which testifies to the predictability of the mixture toxicity of the NBDs.

Molecular Mechanism of Novel Deletions in TPM3 that cause a Hypercontractile Phenotype with Congenital Muscle Stiffness

Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2+-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case.The atomic resolution structure of tropomyosin bound to actin in the ‘switched off’ state shows that tropomyosin makes contact with actin at only two points: one of which is a cluster of basic amino acids: actin K326, K328 and R147. We have demonstrated that two Tpm2.2 mutations, ΔE139 and E181K, and the actin K326N mutation destabilize this actin-tropomyosin interface. We predicted that equivalent charge loss mutations at Tpm3.12 EE 218-219, EE 224-224, or ED 257-258 would also destabilise the interaction with actin leading to a partial switch-on of the muscle. The two newly discovered stiff patient mutation are located at two of the three predicted gain of function sites.We used the quantitative in vitro motility assay and skeletal muscle thin filaments containing recombinant mutant Tpm3.12 expressed in a Baculovirus/sf9 system. ΔE218 led to a 2.5-fold increase in Ca2+-sensitivity (EC50 ratio ΔE218/WT = 0.40 ± 0.07, ). ΔE224 also showed an increase in Ca2+-sensitivity by 2.2-fold (EC50 ratio ΔE224/WT for = 0.46 ± 0.09). It has been previously shown that there was a 2.5 fold increase in Ca2+ sensitivity for ACTC K326N mutation (EC50 ratio K326N/WT = 0.4 ± 0.05, p=0.07).The increased Ca2+-sensitivity indicates that both mutations cause a gain of function that was predicted from the structural analysis and that can account for the stiff patient syndrome.

Design and Synthesis of Non-Peptide, Selective Orexin Receptor 2 Agonists.

Orexins are a family of neuropeptides that regulate sleep/wakefulness, acting on two G-protein-coupled receptors, orexin receptors 1 (OX1R) and 2 (OX2R). Genetic and pharmacologic evidence suggests that orexin receptor agonists, especially OX2R agonist, will be useful for mechanistic therapy of the sleep disorder narcolepsy/cataplexy. We herein report the discovery of a potent (EC50 on OX2R is 0.023 μM) and OX2R-selective (OX1R/OX2R EC50 ratio is 70) agonist, 4'-methoxy-N,N-dimethyl-3'-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl)sulfamoyl]-(1,1'-biphenyl)-3-carboxamide 26.

Two-stage prediction of the effects of imidazolium and pyridinium ionic liquid mixtures on luciferase.

The predicted toxicity of mixtures of imidazolium and pyridinium ionic liquids (ILs) in the ratios of their EC50, EC10, and NOEC (no observed effect concentration) were compared to the observed toxicity of these mixtures on luciferase. The toxicities of EC50 ratio mixture can be effectively predicted by two-stage prediction (TSP) method, but were overestimated by the concentration addition (CA) model and underestimated by the independent action (IA) model. The toxicities of EC10 ratio mixtures can be basically predicted by TSP and CA, but were underestimated by IA. The toxicities of NOEC ratio mixtures can be predicted by TSP and CA in a certain concentration range, but were underestimated by IA. Our results support the use of TSP as a default approach for predicting the combined effect of different types of ILs at the molecular level. In addition, mixtures of ILs mixed at NOEC and EC10 could cause significant effects of 64.1% and 97.7%, respectively. Therefore, we should pay high attention to the combined effects in mixture risk assessment.

Prediction of Antiplatelet Effects of Aspirin In Vivo Based on In Vitro Results

The aim of this study was to establish a method to predict the antiplatelet effects of aspirin in vivo based on in vitro results. Aspirin in 5 different concentrations was added to the platelet-rich plasma samples, and the rates of platelet aggregation induced by collagen were determined in vitro. In addition, platelet aggregation and plasma drug concentration values were determined in vivo before and after the administration of aspirin (162 mg). The 50% effective concentration (EC50) values obtained from the in vivo and in vitro experiments were shown to have relevance, because the EC50 ratio for each subject was the same (0.23 ± 0.03). The actual and predicted values for the rate of inhibition of platelet aggregation were well correlated (P < .0001, r = .95) when the predicted rate was determined using the present method. Our results suggest that the antiplatelet effects of aspirin can be predicted using blood samples obtained before its administration.

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.

BAL27862: A unique microtubule-targeted agent that severs microtubules and overcomes multifactorial drug resistance.

e13589 Background: Microtubule-targeted agents (MTAs) are widely used to treat human malignancies. A major limitation of their efficacy is the development of resistance modulated by P-gp overexpression or tubulin and/or apoptotic pathway protein modifications. BAL27862 is a novel MTA that triggers apoptosis in cancer cells. In vitro, BAL27862 destabilizes microtubules to produce a microtubule phenotype distinct from that observed with other MTAs, and retains its antiproliferative activity against P-gp overexpressing tumor cells. BAL27862, when administered intravenously or orally, induces significant antitumor responses in a range of animal models of human cancer, including tumors refractory to conventional treatments. Methods: First, we compare the in vitro activity of BAL27862 with conventional MTAs paclitaxel (PTX) and vinblastine (VLB). The antiproliferative potency of BAL27862 was determined in a PTX- and VLB-resistant ovarian cancer cell line. Resistance was mediated by P-gp over-expression, Bcl-2 down-regulation and tubulin modifications. We then analyzed the effects of BAL27862 on microtubule dynamics (MDs) in SKBr3 breast cancer cells compared with PTX. Results: BAL27862 remained fully active against PTX- and VLB-resistant cells when compared with the chemosensitive parental line. The resistance factors observed (defined as EC50 ratio of resistant vs. parental cells) were 1.14 (NS), 147 (p<0.01) and 57 (p<0.01) for BAL27862, PTX and VLB respectively. These results confirm that BAL27862 overcomes P-gp overexpression-mediated resistance, and demonstrate that Bcl-2 status and tubulin modifications do not necessarily affect its antitumor activity. Analysis of MDs shows that BAL27862 elicited effects consistent with its destabilizing activity; suppression of MDs was 2-fold lower than observed with PTX. Strikingly, BAL27862 displayed a unique microtubule severing activity. Conclusions: Besides its microtubule-targeting activity, BAL27862 displays unique features in its mechanism of action that make it a promising compound for clinical investigation. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Basilea Pharmaceutica Basilea Pharmaceutica

Is the clinical efficacy of epidural diamorphine concentration-dependent when used as analgesia for labour?

The physicochemical properties of diamorphine (3,6-diacetylmorphine) enhance its bioavailability compared with more lipid-soluble opioids when administered into the epidural space. However, the influence of concentration, volume or mass on the clinical efficacy of diamorphine is not known. In this double-blind, randomized, prospective study, 62 women in active labour and </=5 cm cervical dilatation were recruited to determine whether the mode of action of diamorphine in the epidural space is concentration-dependent. After insertion of a lumbar epidural catheter, patients received epidural diamorphine 3 mg either as a high-volume, low-concentration solution (group A) or a low-volume, high-concentration solution (group B). The concentration of diamorphine was determined by the response of the previous patient in the same group using up-down sequential allocation. Pain corresponding to the previous contraction was assessed using a 100-mm visual analogue score and effective analgesia was defined as </=10 mm within 30 min of epidural injection. There was no significant difference in EC50 for diamorphine between the groups: the difference was 15.0 microg ml(-1) (95% CI -40.3 to 10.3). The EC50 for group A was 237.5 microg ml(-1) (95% CI 221.2 to 253.8) and the EC50 for group B was 252.5 microg ml(-1) (95% CI 232.2 to 272.8). The EC50 ratio was 0.95 (95% CI 0.87 to 1.06). The groups exhibited parallelism (P=0.98). The overall EC50 for all data was 244.2 microg ml(-1) (95% CI 230.8 to 257.2). We conclude that diamorphine provides analgesia in labour by a concentration-dependent effect.

Toxicity of cadmium, endosulfan, and atrazine in adrenal steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana.

The effects of cadmium, endosulfan, and atrazine on corticosterone secretion and viability of adrenal cells of African clawed frog (Xenopus laevis) and bullfrog (Rana catesbeiana) were assessed in vitro using a new bioassay. The bioassay relies on stimulation with adrenocorticotropic hormone (ACTH), the endogenous secretagogue for corticosterone secretion, and with dibutyryl cyclic adenosine monophosphate (dbcAMP), an analogue of cAMP, to pinpoint the site of action of the xenobiotics within the steroidogenic cell. To compare the test toxicants according to their endocrine-disrupting potential, the lethal concentration needed to kill 50% of the cells:effective concentration of 50% (LC50:EC50) ratio was calculated, with LC50 as the concentration that kills 50% of the steroidogenic cells and the EC50 as the concentration that impairs corticosterone secretion by 50%. The higher the ratio, the higher the potential for endocrine disruption. Atrazine had no affect on cell viability and on corticosterone secretion in X. laevis, but its endocrine-disrupting potential was high in R. catesbeiana. The LC50:EC50 ratio for cadmium and endosulfan in X. laevis was 26.07 and 1.23, respectively, and for atrazine, cadmium, and endosulfan in R. catesbeiana it was 909, 41, and 3, respectively. The dbcAMP did not restore corticosterone secretion in the cells exposed to the test toxicants in both species. Our study suggests that the secretory capacity of adrenal cells of amphibians can be impaired by environmental chemicals, especially atrazine in the bullfrog, and that these adrenotoxicants disrupt the enzymatic pathways leading to corticosterone secretion downstream from the step-generating cAMP.

Cytochrome P4501 a induction in avian hepatocyte cultures: A promising approach for predicting the sensitivity of avian species to toxic effects of halogenated aromatic hydrocarbons

Concentration-dependent effects of halogenated aromatic hydrocarbons(HAHs) on cytochrome P4501A (CYP1A) induction in primary hepatocyte cultures prepared from embryos of chickens (four breeds), pheasants, turkeys, ducks (three breeds), and herring gulls were determined. CYP1A activity was estimated by measuring ethoxyresorufin O-deethylase (EROD) activity and the concentration of immunodetectable CYP1A was estimated using mouse monoclonal antibody 1-12-3 that was prepared against scup (Stenotomus chrysops) CYP1A1. The HAHs studied were 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 3,4,4′,5-tetrachlorobiphenyl (PCB 81), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105), and 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118). Two general types of comparisons were made: (1) relative potencies of compounds within a species (expressed relative to TCDD as induction equivalency factors, IEFs) and (2) relative sensitivity of each species to EROD induction by each compound. Three methods for estimating potency were compared. These were: (1) the concentration of inducer that produced a half-maximal (EC50) EROD response, (2) the concentration producing a response equivalent to 10% of the maximal response produced by TCDD (ECTCDD10%), and (3) a slope ratio method. For each method, the rank order in potency was TCDD ⩾ TCDF > PCB 126 > PCB 81 > PCB 77 > PCB 169 in chicken, pheasant, and turkey hepatocytes. The rank order was similar in duck and herring gull hepatocytes with the following exceptions: TCDF was approximately 2- to 4-fold more potent than TCDD in duck hepatocytes; PCB 169 induced EROD in gulls, but PCB 77 had no measurable effect in this species. PCB 118 was a relatively weak EROD inducer in most species/breeds, but it did not induce EROD in Pekin ducks or gulls. PCB 105 was a weak inducer in White Leghorn chicken and turkey hepatocytes, but it did not induce EROD in other species. The EC50, ECTCDD10% and slope ratio methods for estimating potencies generally gave similar IEFs for compounds that produced a maximal response that was at least 60% of the maximal response produced by TCDD. For compounds that caused a response that was 50% or lower than that produced by TCDD, EC50-based IEFs were greater (10- to 100-fold) than ECTCDD10%-based IEFs or slope-ratio-based IEFs. Among species, the rank order in sensitivity to EROD induction was chicken > pheasant > turkey ⩾ duck ⩾ herring gull. The relative sensitivity of avian hepatocyte cultures to EROD induction by PCB 77 was similar to the relative sensitivity of these species (reported elsewhere) to lethality after in ovo injection of PCB 77. Chicken hepatocyte cultures were 5–10 times more sensitive to EROD induction by TCDD than were pheasant hepatocyte cultures, which is identical to the difference in sensitivity of these species to the lethal effect of TCDD after in ovo injection. Measuring the sensitivity of hepatocyte cultures to EROD induction might be useful for estimating the sensitivity of avian species (including rare or endangered species, where it is impossible to conduct in vivo studies) to the embryotoxic effects of TCDD, non-ortho substituted PCBs, and other aryl hydrocarbon receptor agonists.

Prediction of the therapeutic dose for β-stimulants based on preclinical data: Application of oral dosage forms and aerosols to asthmatic patients

The therapeutic doses of beta-stimulants in the treatment of bronchial asthma show wide variation among the drugs. In the present study, we tried to construct a system for prediction of the optimum clinical dose of beta-stimulants as oral dosage forms and aerosols using the data from preclinical pharmacological studies and pharmacokinetic data in humans. Values of EC50 (drug concentration that gives the half-maximum bronchodilator effect) in the in vitro studies based on isolated trachea of guinea pigs were collected from the literature. The ratios of the EC50 value of each drug to that of isoproterenol were used as the indicator of in vitro pharmacological activity. There were significant correlations between the EC50 ratios and the oral dose or the maximum plasma concentration after single oral administration, but the correlation coefficients were relatively small (r < .9). On the other hand, a linear log-log relationship was observed between the EC50 ratio and the maximum plasma unbound concentration (Cu(max)) (slope = 0.907, r = .955, p < 0.001). In the case of aerosols, a good correlation was observed between the EC50 ratio and the dose (slope = 0.770, r = .900, p < 0.01). The predicted concentration of beta-stimulants in the lung after aerosol administration tended to be higher than the Cu(max) after oral administration, suggesting the contribution of nonspecific binding in the lung tissue. These findings indicate the possibility of predicting the appropriate dose of beta-stimulants based on the preclinical pharmacological data and the pharmacokinetic data in humans.

Microtesting appraisal of ATP and cell recovery toxicity end points after acute exposure of Selenastrum capricornutum to selected chemicals

AbstractEmploying microtesting procedures, intracellular ATP and cell recovery (96‐h cell count determinations) were appraised as toxicity end‐point responses after acute (4‐h) exposure of the green alga Selenastrum capricornutum to metals (Cu2+, Co2+, Cd2‐, Cr3+, Cr6+, Ni2+, Hg2+, Zn2+, B3+), sodium azide, and 4 organic compounds (sodium dodecyl sulphate, 2,4‐dichlorophenol, phenol, tebuthiuron). Except for B3‐, both end points were able to measure acute metal phytotoxicity effects at concentrations of 5 mg · L−1 or lower. The algal recovery test showed better metal toxicity discrimination than the ATP test on the basis of dose—response slope steepness and elevation differences. Both tests, however, supplied useful complementary information. While the ATP test reacted rapidly to chemical aggression, the recovery test offered pertinent insight into actual long‐term consequences of acute exposure. With the nonmetals, both end‐point responses were mitigated (sodium azide, sodium dodecyl sulphate, 2,4‐dichlorophenol) or insensitive (phenol, tebuthiuron), suggesting low acute exposure risk to algae by these chemicals. Based on 4‐h (ATP and cell recovery) EC50 to standard 96‐h EC50 ratio values, the algal indicator was shown to have low tolerance to 6 chemicals (Cr3+, Cr6+, Hg2‐, Cu2+, sodium dodecyl sulphate, 2,4‐dichlorophenol). When compared to acute exposure bacterial assays, the acute exposure ATP and cell recovery microtests competed well in terms of overall sensitivity to a selection of chemicals. In general, this work demonstrates the utility that short‐term algal exposure tests can provide for chemical toxicity investigations.