Sensory irritation mechanisms investigated from model compounds: trifluoroethanol,hexafluoroisopropanol and methyl hexafluoroisopropyl ether

Quantitative structure-activity relationships (QSAR) have suggested the importance of hydrogen bonding in relation to activation of the sensory irritant receptor by nonreactive volatile organic chemicals. To investigate this possibility further, three model compounds with different hydrogen bond acidity, trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether, were selected for study. The potency of each chemical is obtained from the concentration necessary to reduce respiratory rate in mice by 50% (RD50). The RD50 values obtained were: methyl hexafluoroisopropyl ether (?160?000?ppm), trifluoroethanol (11?400–23?300?ppm), and hexafluoroisopropanol (165?ppm). QSAR showed that trifluoroethanol and methyl hexafluoroisopropyl ether behaved as predicted as nonreactive sensory irritants, whereas hexafluoroisopropanol was much more potent than predicted. The higher than predicted potency of hexafluoroisopropanol could be due to a coupled reaction, involving both strong hydrogen bonding and weak Brönsted acidity. A concerted reaction could thus be more efficient in activation of the receptor. Hydrogen bonding properties and concerted reactions may be important in the activation of the sensory irritant receptor by nonreactive volatile organic chemicals.     

Glutathione depletion in rat hepatocytes: a mixture toxicity study with α,β-unsaturated esters

1. Glutathione (GSH) depletion is often reported as an early cytotoxic effect, caused by many reactive organic chemicals. In the present study, GSH depletion in primary rat hepatocytes was used as an in vitro effect-equivalent to measure the toxic potency of α,β-unsaturated esters (acrylates and methacrylates). 2. When these compounds were administered as a mixture, GSH depletion was dose additive. The result of the mixture study shows that GSH depletion may be a useful effect-equivalent for the risk assessment of mixtures of α,β-unsaturated esters. 3. To get more insight in the underlying mechanisms of GSH depletion, the metabolism of two esters was investigated in greater detail. One of them, allyl methacrylate, was metabolized to acrolein. This metabolic pathway can explain the high potency of allyl methacrylate to deplete GSH despite its low intrinsic chemical reactivity.     

A new in vitro method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells

Allergic contact dermatitis (ACD) is a significant safety concern for developers of cosmetic, personal care, chemical, pharmaceutical, and medical device products. The guinea pig maximization test (GMPT) and the murine local lymph node assay (LLNA) are accepted methods for determining chemical sensitization. Recent legislative initiatives in Europe require the development of new in vitro alternatives to animal tests for chemical sensitization. The aim of this project was to develop an in vitro screening method that uses a human skin cell line (HaCaT), chemical reactivity, and gene expression profiling to identify positive and negative responses, to place chemicals into potency categories of extreme/strong (ES), moderate (M), weak (W), and nonsensitizers (N), and to provide an estimate of corresponding LLNA values. The method and processing algorithm were developed from a training set of 39 chemicals possessing a wide range of sensitization potencies. Three cationic metals, chromium (Cr), nickel (Ni), and silver (Ag), were also evaluated in this model. Chemical reactivity was determined by measuring glutathione (GSH) depletion in a cell free matrix. Three signaling pathways (Keap1/Nrf?2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE) that are known to be activated by sensitizing agents were monitored by measuring the relative abundance of 11 genes whose expression is controlled by one of these 3 pathways. Final exposure concentrations were based on toxicity and solubility. A range-finding experiment was conducted with each compound to determine cytotoxicity and solubility. Six exposure concentrations (0.1 to 2,500?μM) and an exposure time of 24 hours were used in the final experiments. Glutathione depletion alone did not provide the accuracy necessary to differentiate potency categories. However, chemical reactivity combined with gene expression profiles significantly improved the in vitro predictions. A predicted toxicity index (PTI) was determined for each test chemical. A comparison of LLNA values with PTI values revealed an inverse relationship. The large variation in LLNA data for compounds in the same potency category makes direct extrapolation from PTI to LLNA difficult. To challenge the system, 58 additional compounds were submitted in a blinded manner. Compounds placed into ES and M categories were considered positive, whereas compounds classified as W or N were considered negative. Accuracy was approximately 84%, with a sensitivity of 81% and a specificity of 92%. The model correctly identified 2 of 3 cationic metals as positive. In conclusion, the method described here demonstrates a valuable in vitro method for identifying chemicals and metals that induce skin sensitization.     

A quantitative structure—activity relationship study and ecotoxicological risk quotient for the protection from chemical pollution

A quantitative structure—activity relationship study is carried out with use of the toxicity (LC₅₀) of the various kinds of fish obtained from the references. The equation obtained contained the parameters of molecular weight (MW) and mean ionization potential (IP ):log(1/LC₅₀) = 28.665 + 0.01095MW - 2.570IP (R² = 0.695, n = 581). This equation could estimate the toxicity of the chemicals detected in the environment of Japan by Environment Agency (r = 0.80, n = 53). In order to evaluate the hazardousness, we propose the Ecotoxicological Risk Quotient (ERQ): ERQ^c = -log Concentration in the environment/Effective concentration in the test ERQ^a = -log Σ Concentration in the environment/Effective concentration in the test ERQ^c and ERQ^a are the indices of the hazardousness of a chemical and chemicals in an area, respectively. ERQ could explain the status of pollution in water environment of Japan.     

Integrated testing and assessment approaches for skin sensitization: a commentary

A Bayesian integrated testing strategy (ITS) approach, aiming to assess skin sensitization potency, has been presented, in which data from various types of in vitro assays are integrated and assessed in combination for their ability to predict in vivo skin sensitization data. Here we discuss this approach and compare it to our quantitative mechanistic modeling (QMM) approach based on physical organic chemistry. The main findings of the Bayesian study are consistent with our chemistry‐based approach and our previously published assessment of the key determinants of sensitization potency, in particular the relatively high predictive value found for chemical reactivity data and the relatively low predictive value for bioavailability parameters. As it stands at present the Bayesian approach does not utilize the full range of predictive capability that is already available, and aims only to assign potency categories rather than numerical potency values per se. In contrast, for many chemicals the QMM approach can already provide numerical potency predictions. However, the Bayesian approach may have potential for those chemicals where a chemistry modeling approach cannot provide a complete answer (e.g. pro‐electrophiles whose in cutaneo activation cannot currently be modeled confidently). Nonetheless, our main message is of the importance of leveraging chemistry insights and read‐across approaches to the fullest extent possible. Copyright © 2013 John Wiley & Sons, Ltd.     

Empirical model of mesothelioma potency factors for different mineral fibers based on their chemical composition and dimensionality

Context: The potency of various mineral fiber types to produce mesothelioma was previously evaluated for numerous cohorts, but the differences in potencies for distinct fiber types have yet to be explained.

Objective: To develop an empirical model that would reconstruct mesothelioma potency factors for various types of fiber based on their chemical composition and dimensionality.

Methods: Typical chemical composition and dimensionality metrics (aspect ratios) were obtained and combined with mesothelioma potency factors estimated by Hodgson and Darnton method for Quebec chrysotile, South Africa amosite, South Africa and Australian crocidolite, Russian anthophyllite, Libby amphiboles, and Turkey erionite. The forward stepwise log–log regression method was utilized to determine the best combination of input parameters.

Results: Mesothelioma potency factors (R_M) for selected cohorts were effectively reconstructed utilizing the median aspect ratio of fibers and equivalent fractions of SiO₂, total Fe oxides or total equivalent Fe³⁺ as Fe₂O₃, and MgO. Modeled potency factors increase as the aspect ratio, SiO₂, and total Fe oxide (or Fe₂O₃) content grow, and as the MgO content diminishes. Correlation coefficients up to 0.999, p?<?0.01, were achieved. The models also yield reasonable estimates of mesothelioma potencies for other fiber types, including Bolivian crocidolite, Russian chrysotile, fluoro-edenite, and others.

Conclusion: In spite of the empirical approach, the proposed models provide a starting point for targeted studies of mesothelioma mechanisms by elucidating significant contributing physicochemical factors. The models have an exploratory and preliminary character but can potentially be useful to introduce quantitative structure–activity relationship approaches for the toxicology of fibrous minerals.     

Toxic Equivalency Factors of Polybrominated Dibenzo-p-dioxin, Dibenzofuran, Biphenyl, and Polyhalogenated Diphenyl Ether Congeners Based on Rainbow Trout Early Life Stage Mortality

Polybrominated and polychlorinated biphenyls (PBBs/PCBs), dibenzo-p-dioxins (PBDDs/PCDDs), dibenzofurans (PBDFs/PCDFs), and diphenyl ethers (PBDEs/PCDEs) are persistent, lipophilic environmental contaminants that may pose a risk to fish early life stage survival. To determine this potential risk, a rainbow trout early life stage mortality bioassay was used in which the potency of individual polybrominated chemicals was compared to the potency of the most potent polychlorinated chemical in these classes, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Following injection of newly fertilized rainbow trout eggs, fish-specific toxic equivalency factors (TEFs) were calculated as the molar ratio of TCDD LD50 to brominated compound LD50. Signs of toxicity were identical to those produced by polychlorinated TCDD-like chemicals and included yolk sac edema, pericardial edema, multifocal hemorrhages, reduced growth, and craniofacial malformations. Polybrominated dibenzo-p-dioxins, dibenzofurans, and biphenyls exhibited decreased potency with increased bromine substitution. Only 2,3,7,8-TBDD was more potent than 2,3,7,8-TCDD, whereas other polybrominated dibenzo-p-dioxins were equipotent or less potent than identically substituted polychlorinated dibenzo-p-dioxins in this assay. Although two PBDF congeners were equipotent to identically substituted PCDFs, 2,3,7,8-TBDF was 9-fold more potent than 2,3,7,8-TCDF. Both 3,3′,4,4′-TBB and 3,3′,4,4′,5,5′-HxBB were 10-fold more potent than identically substituted polychlorinated biphenyls. The halogenated diphenyl ethers and di-ortho polybrominated biphenyls were inactive in this assay. Thus, in thisin vivoassay the polybrominated and polychlorinated TCDD-like chemicals were not always equally potent. To assess the risk posed by mixtures of these chemicals to feral fish populations, fish-specific TEFs for both polybrominated and polychlorinated chemicals should be used.     

Simple physicochemical properties related with lipophilicity,polarity, molecular size and ionization status exert significant impact on the transfer of drugs and chemicals into human breast milk

Objectives: The transfer of xenobiotic compounds into human breast milk has raised serious concerns in the last few years. The present study is aimed to assess whether simple physicochemical properties exert significant impact on human breast milk transfer of drugs and chemicals.

Methods: A large data set of 375 xenobiotic compounds with available experimental milk to plasma (M/P) ratios was systematically compiled from the literature and explored with their physicochemical properties being further analyzed with respect to their extent to transfer into breast milk.

Results: Xenobiotic compounds with increased breast milk transfer (M/P ≥ 1) were characterized by enhanced lipophilicity and decreased molecular size (p < 0.05). Enhanced polarity and hydrogen bonding capacity were more frequently observed in xenobiotic compounds with reduced breast milk transfer (p < 0.0001). Xenobiotic compounds presenting increased positive charge at pH 7.4 were characterized by enhanced breast milk transfer (p < 0.001). Xenobiotic compounds presenting increased negative charge at pH 7.4 were characterized by decreased breast milk transfer (p < 0.001).

Conclusions: The present study supports evidence that simple physicochemical properties related with lipophilicity, polarity, molecular size and ionization status exert significant impact on drugs and chemicals transport into human breast milk.     

Development of a Generalized,Quantitative Physicochemical Model of CYP3A4 Inhibition for Use in Early Drug Discovery

Purpose. To examine the structure–activity relationships for the inhibition of the activity of recombinant human CYP3A4 and to establish a generalized, quantitative physicochemical model for use in early drug discovery. Methods. Inhibition of the activity of recombinant human CYP3A4 (erythromycin N–demethylase) by 30 diverse chemicals was studied using enhanced throughput methodology. Results. There was a general, strong correlation between the IC₅₀ value determined against erythromycin N–demethylase activity and lipophilicity (LogD_7.4) (r ² = 0.68, p <0.0001). This relationship was strengthened further by subdividing the structures studied into two distinct subpopulations of chemistry within the dataset. These could be identified by the absence (r ² = 0.80, p <0.0001) or presence (r ² = 0.69, p <0.0001) of a sterically uninhindered N–containing heterocycle, more specifically a pyridine, imidazole, or triazole function. The presence of these structural motifs increased the potency of CYP3A4 inhibition by approximately 10–fold for a given lipophilicity (LogD_7.4.value). More detailed analyses of AstraZeneca compounds demonstrated that the inhibitory potency of the pyridine structure can be attenuated through direct steric effects or electronic substitution resulting in a modulation of the pKa of the pyridine nitrogen, thereby influencing its ability to interact with the CYP heme. Conclusions. A generalized, quantitative model is proposed for the inhibition of the major drug metabolizing enzyme, CYP3A4. This model indicates the importance of lipophilicity and rationalizes increased potency arising through additional interactions with the heme iron. These general relationships were shown to be applicable to a selection of compounds of interest to several early research projects.     

Effective exposure of chemicals in in vitro cell systems: A review of chemical distribution models

Nominal effect concentrations from in vitro toxicity assays may lead to inaccurate estimations of in vivo toxic doses because the nominal concentration poorly reflects the concentration at the molecular target in cells in vitro, which is responsible for initiating effects and can be referred to as the biologically effective dose. Chemicals can differentially distribute between in vitro assay compartments, including serum constituents in exposure medium, microtitre plate plastic, headspace and extracellular matrices. The partitioning of test chemicals to these extracellular compartments reduces the concentration at the molecular target. Free concentrations in medium and cell-associated concentrations are considered better proxies of the biologically effective dose. This paper reviews the mechanisms by which test chemicals distribute between in vitro assay compartments, and also lists the physicochemical properties driving the extent of this distribution. The mechanisms and physicochemical properties driving the distribution of test chemical in vitro help explain the makeup of mass balance models that estimate free concentrations and cell-associated concentrations in in vitro toxicity assays. A thorough understanding of the distribution processes and assumptions underlying these mass balance models helps define chemical and biological applicability domains of individual models, as well as provide a perspective on how to improve model predictivity and quantitative in vitro-in vivo extrapolations.     

Skin sensitization: modeling based on skin metabolism simulation and formation of protein conjugates

A quantitative structure-activity relationship (QSAR) system for estimating skin sensitization potency has been developed that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins. A training set of diverse chemicals was compiled and their skin sensitization potency assigned to one of three classes. These three classes were, significant, weak, or nonsensitizing. Because skin sensitization potential depends upon the ability of chemicals to react with skin proteins either directly or after appropriate metabolism, a metabolic simulator was constructed to mimic the enzyme activation of chemicals in the skin. This simulator contains 203 hierarchically ordered spontaneous and enzyme controlled reactions. Phase I and phase II metabolism were simulated by using 102 and 9 principal transformations, respectively. The covalent interactions of chemicals and their metabolites with skin proteins were described by 83 reactions that fall within 39 alerting groups. The SAR/QSAR system developed was able to correctly classify about 80% of the chemicals with significant sensitizing effect and 72% of nonsensitizing chemicals. For some alerting groups, three-dimensional (3D)-QSARs were developed to describe the multiplicity of physicochemical, steric, and electronic parameters. These 3D-QSARs, so-called pattern recognition-type models, were applied each time a latent alerting group was identified in a parent chemical or its generated metabolite(s). The concept of the mutual influence amongst atoms in a molecule was used to define the structural domain of the skin sensitization model. The utility of the structural model domain and the predictability of the model were evaluated using sensitization potency data for 96 chemicals not used in the model building. The TIssue MEtabolism Simulator (TIMES) software was used to integrate a skin metabolism simulator and 3D-QSARs to evaluate the reactivity of chemicals thus predicting their likely skin sensitization potency.     

Predicting Skin Permeability

Published permeability coefficient (K _p) data for the transport of a large group of compounds through mammalian epidermis were analyzed by a simple model based upon permeant size [molecular volume (MV) or molecular weight (MW)] and octanol/water partition coefficient (K _oct). The analysis presented is a facile means to predict the percutaneous flux of pharmacological and toxic compounds solely on the basis of their physicochemical properties. Furthermore, the derived parameters of the model have assignable biophysical significance, and they provide insight into the mechanism of molecular transport through the stratum corneum (SC). For the very diverse group of chemicals considered, the results demonstrate that SC intercellular lipid properties alone are sufficient to account for the dependence of K _p upon MV (or MW) and K _oct. It is found that the existence of an aqueous-polar (pore) pathway across the SC is not necessary to explain the K _p values of small, polar nonelectrolytes. Rather, their small size, and consequently high diffusivity, accounts for their apparently larger-than-expected K _p. Finally, despite the size and breadth of the data set (more than 90 compounds with MW ranging from 18 to >750, and log K _oct ranging from –3 to + 6), the postulated upper limiting value of K _p for permeants of very high lipophilicity cannot be determined. However, the analysis is able to define the physicochemical characteristics of molecules which should exhibit these maximal K _p values. Overall, then, we present a facile interpretation of a considerable body of skin permeability measurements that (a) very adequately describes the dependence of K _p upon permeant size and lipophilicity, (b) generates parameters of considerable physicochemical and mechanistic relevance, and (c) implies that the SC lipids alone can fully characterize the barrier properties of mammalian skin.     

Shape matters: The application of activity-based in vitro bioassays and chiral profiling to the pharmacological evaluation of synthetic cannabinoid receptor agonists in drug-infused papers seized in prisons

Synthetic cannabinoid receptor agonists (SCRAs) elicit many of their psychoactive effects via type-1 human cannabinoid (CB₁) receptors. Enantiomer pairs of eight tert-leucinate or valinate indole- and indazole-3-carboxamide SCRAs were synthesized and their CB₁ potency and efficacy assessed using an in vitro β-arrestin recruitment assay in a HEK239T stable cell system. A chiral high-performance liquid chromatography method with photodiode array and/or quadrupole time-of-flight-mass spectrometry detection (HPLC-PDA and HPLC-PDA-QToF-MS) was applied to 177 SCRA-infused paper samples seized in Scottish prisons between 2018 and 2020. In most samples, SCRAs were almost enantiopure (S)-enantiomer (>98% of total chromatographic peak area), although in some (n = 18), 2% to 16% of the (R)-enantiomer was detected. (S)-enantiomers are consistently more potent than (R)-enantiomers and often more efficacious. The importance of SCRA-CB₁ receptor interactions in the “head” or “linked group” moiety is demonstrated, with the conformation of the “bulky” tert-leucinate group greatly affecting potency (by up to a factor of 374), significantly greater than the difference observed between valinate SCRA enantiomers. (S)-MDMB-4en-PINACA, (S)-4F-MDMB-BINACA, and (S)-5F-MDMB-PICA are currently the most prevalent SCRAs in Scottish prisons, and all have similar high potency (EC₅₀, 1–5 nM) and efficacy. Infused paper samples were compared using estimated intrinsic efficacy at the CB₁ receptor (EIE_CB1) to evaluate samples with variable SCRA content. Given their similar potency and efficacy, any variation in CB₁ receptor-mediated psychoactive effects are likely to derive from variation in dose, mode of use, pharmacokinetic differences, and individual factors affecting the user, rather than differences in the specific SCRA present.