Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro–in silico approach

AbstractDevelopmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro–in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration–response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose–response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose–response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro–in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

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Cd2 Sets Quantitative Thresholds in T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.190.10.1383 ◽

1999 ◽

Vol 190 (10) ◽

pp. 1383-1392 ◽

Cited By ~ 84

Author(s):

Martin F. Bachmann ◽

Marijke Barner ◽

Manfred Kopf

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Dose Response ◽

Response Curve ◽

Cell Activation ◽

Dose Response Curve ◽

Lymphocyte Function

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell–antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose–response curve in vitro by a factor of 3–10. In comparison, stimulation of T cells in the absence of lymphocyte function–associated antigen (LFA)-1–intercellular adhesion molecule (ICAM)-1 interaction shifted the dose–response curve by a factor of 10, whereas absence of both CD2–CD48 and LFA-1–ICAM-1 interactions shifted the response by a factor of ∼100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca2+ fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

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Adenosine preconditions against endothelin-induced constriction of coronary arterioles

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.6.h2593 ◽

2000 ◽

Vol 279 (6) ◽

pp. H2593-H2597 ◽

Cited By ~ 8

Author(s):

Daphne Merkus ◽

David W. Stepp ◽

Deron W. Jones ◽

Yasuhiro Nishikawa ◽

William M. Chilian

Keyword(s):

Ischemic Preconditioning ◽

Dose Response ◽

Response Curve ◽

Protective Function ◽

Vasodilatory Effect ◽

Arteriolar Diameter ◽

Myocardial Hypoperfusion ◽

Coronary Arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng · kg−1 · min−1administered intracoronary, n = 10) induced progressive constriction over 45 min [25 ± 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 μg · kg−1 · min−1administered intracoronary) for 20 min and 10 min of washout ( n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 ± 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50–150 μm) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM–5 nM) was rightward shifted after preconditioning with adenosine (1 μM) for 20 min and 10 min of washout ( n = 11). Blockade of A2 receptors [8-(3-chlorostyryl)caffeine, 1 μM, n = 9] but not A1 receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A2 receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.

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ETUDE DE L'ACTION DU FACTEUR HYPOTHALAMIQUE LRF (LH-RELEASING FACTOR) CHEZ LE RAT IN VIVO & IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.0550481 ◽

1967 ◽

Vol 55 (3) ◽

pp. 481-496 ◽

Cited By ~ 16

Author(s):

Marian Jutisz ◽

Annette Bérault ◽

Marie-Anne Novella ◽

Geneviève Ribot

Keyword(s):

Dose Response ◽

Crude Extract ◽

Response Curve ◽

Assay Method ◽

Hypothalamic Extract ◽

Rat Pituitary ◽

Pituitary Glands ◽

Releasing Effect

ABSTRACT A highly purified ovine LH-releasing factor (LRF) was obtained by a modification of the method previously described. After the fractionation of a crude hypothalamic extract on a Sephadex G-25 column, the LRF fraction was desalted and partially purified by chromatography on a Dowex 50 × 12 column and on an Amberlite CG 4B column. The last step of this method, chromatography on a CMC column, gave a purification of about 1600 times with respect to the crude extract. The action of this highly purified LRF preparation was studied on rat pituitary glands in vivo and in vitro. The method used in vivo was the evaluation of the LH-releasing effect of LRF in chronically ovariectomized, steroid-blocked rats (Ramirez & McCann 1963 b). A procedure was developed which allows a 4-fold concentration of the plasma LH from these rats, so that it can be assayed by a 4-point assay method. In the in vitro method, the pituitary glands of ovariectomized steroidblocked rats (Schally & Bowers 1964 a) were incubated in a Krebs-Ringer buffer with or without LRF, and the LH released into the medium was assayed using the O.A. A.D. method of Parlow. A dose-response curve was established between the log doses of LRF and the amount of LH released. This method can be used as a sensitive and specific assay for LRF. It was shown that a dose of 1.22 μg of LRF releases approximately 5 μg of LH per mg of pituitary tissue. This is about double of the amount of this hormone originally present in the pituitary glands of these rats (2.7 μg/mg). This leads us to the conclusion that the excess of this hormone was probably synthetized during the process of incubation. The amount of steroids injected as a blocking agents, appears to be very important for both in vivo and in vitro tests.

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Prediction of dose-dependent in vivo acetylcholinesterase inhibition by profenofos in rats and humans using physiologically based kinetic (PBK) modeling-facilitated reverse dosimetry

Archives of Toxicology ◽

10.1007/s00204-021-03004-4 ◽

2021 ◽

Vol 95 (4) ◽

pp. 1287-1301

Author(s):

Isaac Omwenga ◽

Shensheng Zhao ◽

Laetitia Kanja ◽

Hans Mol ◽

Ivonne M. C. M. Rietjens ◽

...

Keyword(s):

Dose Response ◽

In Silico ◽

Acetylcholinesterase Inhibition ◽

Ache Inhibition ◽

Response Data ◽

Physiologically Based ◽

Parameter Values ◽

Dose Dependent

AbstractOrganophosphate pesticides (OPs) are known to inhibit acetylcholine esterase (AChE), a critical effect used to establish health-based guidance values. This study developed a combined in vitro–in silico approach to predict AChE inhibition by the OP profenofos in rats and humans. A physiologically based kinetic (PBK) model was developed for both species. Parameter values for profenofos conversion to 4-bromo-2-chlorophenol (BCP) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on profenofos-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose–response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose–response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Altogether, the results demonstrate that in vivo AChE inhibition upon acute exposure to profenofos was closely predicted in rats, indicating the potential of this novel approach method in chemical hazard assessment.

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Correspondence of In Vitro and In Vivo Fluconazole Dose-Response Curves for Cryptococcus neoformans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.8.3297-3301.2005 ◽

2005 ◽

Vol 49 (8) ◽

pp. 3297-3301 ◽

Cited By ~ 18

Author(s):

Robert A. Larsen ◽

Madeline Bauer ◽

Ann M. Thomas ◽

Alejandro Sanchez ◽

Diane Citron ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Dose Response ◽

Response Curve ◽

Drug Susceptibility Testing ◽

Response Curves ◽

In Vitro Susceptibility ◽

Drug Concentrations ◽

Wide Range

ABSTRACT We conducted in vitro experiments to evaluate the susceptibility of a clinical isolate of Cryptococcus neoformans to a wide range of concentrations of fluconazole. In vitro susceptibility was tested using broth macrodilution methods modified to provide a numeric count of viable organisms. The association between the quantitative in vitro response and fluconazole drug concentrations was estimated using local nonparametric regression. Regression analysis was used to assess the correspondence between the in vitro fluconazole concentration-response curve and the murine dose-response curve observed in our previously reported murine model. The regression model was then used to predict the murine response. There was a strong correspondence between in vitro measures of response to fluconazole alone and the previously reported biologic effects seen in the mouse. In vitro antifungal drug susceptibility testing can reliably predict the murine response to fluconazole.

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856 INBRX-106: a novel hexavalent anti-OX40 agonist for the treatment of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.856 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A897-A897

Author(s):

Emily Rowell ◽

Heather Kinkead ◽

Elisabeth Torretti ◽

Bryan Becklund ◽

Florian Sulzmaier ◽

...

Keyword(s):

T Cell ◽

Dose Response ◽

Response Curve ◽

Single Agent ◽

Cell Assays ◽

Human Igg1 ◽

Anti Tumor Activity ◽

T Cell Assays

BackgroundOX40 is a co-stimulatory receptor enriched on immune cells in the tumor microenvironment. OX40 agonism promotes anti-tumor responses, both singly and in combination with checkpoint inhibitors. The cognate OX40 ligand, OX40L, is a trimeric protein that activates robust signaling through clustering. INBRX-106 is a novel hexavalent OX40 agonist that has been rationally designed to optimize target clustering and provide superior agonism to previously explored bivalent entities, leading to more potent anti-tumor activity.MethodsINBRX-106 is a homodimer, each half comprising three identical humanized, camelid single-domain antibody binding domains targeting OX40 linked end-to-end, and fused to an effector-enabled human IgG1 constant domain (Fc). Due to lack of rodent cross-reactivity, a valency, affinity and activity-matched murine surrogate, Hex-C04, was generated for the purpose of preclinical modeling. Hex-C04 contains an mIgG2a effector enabled Fc, the mouse isotype most analogous to the activity of human IgG1. The activity and potency of INBRX-106 and Hex-C04 were evaluated in functional in vitro T-cell assays, and the anti-tumor efficacy of Hex-C04 was evaluated alone or in combination with PD-1 blockade across a number of syngeneic tumor models.ResultsINBRX-106 binds specifically to OX40 with a sub nanomolar apparent affinity, without blocking the binding of its ligand OX40L. In vitro, cross-linking by INBRX-106 rapidly induces loss of OX40 surface expression in addition to driving receptor signaling. In primary T-cell assays, INBRX-106 is more potent than a bivalent comparator antibody, inducing greater upregulation of activation markers, cytokine production and proliferation. This costimulatory activity exhibits a bell-shaped dose-response curve, with maximal activity occurring at receptor occupancies of 30–100%. In vivo, tumor growth control by Hex-C04 also follows a bell-shaped dose response curve. Rapid loss of OX40 is observed in vivo as well, with both the degree and duration of OX40 loss dependent on Cmax and exposure. Hex-C04 demonstrated strong single-agent activity across a variety of preclinical tumor models including models that do not respond to a PD-1/PD-L1 checkpoint inhibitor, and this activity was improved in combination with a PD-1 blocking antibody.ConclusionsPreclinically, INBRX-106 significantly outperforms bivalent antibodies in co-stimulatory capacity and anti-tumor activity. On the weight of this data, Inhibrx Inc. has initiated a first-in-human Phase 1 trial of INBRX-106 as a single agent or in combination with Keytruda® (pembrolizumab). The complex relationship between dose, OX40 target modulation and activity indicate the importance of integrating preclinical data sets with emerging clinical data to make informed decisions regarding INBRX-106 dose and schedule.Trial RegistrationNCT04198766Ethics ApprovalThe care and use of all animals were reviewed and approved by the IACUC committees of Explora BioLabs and Molecular Diagnostic Services and conducted in accordance with AAALAC regulations.

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Reproductive and developmental toxicity testing: from in vivo to in vitro

ALTEX ◽

10.14573/altex.2012.3.333 ◽

2012 ◽

Author(s):

Elizabeth Stallman Brown

Keyword(s):

Developmental Toxicity ◽

Toxicity Testing ◽

Developmental Toxicity Testing

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A novel in silico tool for dose assessment in cell culture nanotoxicology

Biomedical Science and Engineering ◽

10.4081/bse.2021.156 ◽

2021 ◽

Vol 4 (s1) ◽

Author(s):

Ermes Botte ◽

Pietro Vagaggini ◽

Joana Costa ◽

Lara Faccani ◽

Ilaria Zanoni ◽

...

Keyword(s):

Cell Culture ◽

Dose Response ◽

In Silico ◽

Computational Approach ◽

Engineered Nanomaterials ◽

Dose Assessment ◽

Biological Dose ◽

Toxicity In Vitro

Dose assessment is essential for understanding the mechanisms triggering nanomaterial toxicity in vitro and for meaningful translations to in vivo. We propose a novel computational approach for improving the accuracy of biological dose-response characterization, demonstrating its robustness for insoluble Engineered Nanomaterials (ENMs).

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