In vitro toxicity assessment of nanocrystals in tissue‐type cells and macrophage cells

Nanocrystals (NCs), a type of innovative material particle, are a potential drug delivery platform that aims to improve the bioavailability of hydrophobic drugs. However, due to the lack of consideration of their toxicity, existing studies have not investigated whether the nanoscale properties of NCs, such as particle sizes, may lead to NC‐induced toxicity. Because of the disparity between the rapid development of NCs and the lack of studies regarding NC toxicity, the present study investigated possible NC toxicity and clarified the relationship between particle sizes and NC toxicity. RAW264.7 and HepG2 cells were chosen as representatives of macrophage cells and tissue‐type cells, respectively. Monosodium urate NCs were used as a drug model. Different particle sizes of monosodium urate NCs were prepared using precipitation methods. Methyl tetrazolium, lactate dehydrogenase, oxidative stress and apoptosis/necrosis assays were then used to evaluate cell damage and recovery. The results showed that small NC particle sizes produced higher toxicity than larger ones. In immune cells, these cytotoxic effects were greater than in tissue cells. After removal of small NCs, tissue cell damage could be significantly reversed, while immune cells were only slightly restored. However, after removal of large NCs, both cell types had almost no recovery. In summary, despite conventional wisdom, our research confirmed that NCs are not very safe and that NC particle sizes are closely related to the degree of NC toxicity.     

In vitro investigation of the genotoxic and cytotoxic effects of thiacloprid in cultured human peripheral blood lymphocytes

Thiacloprid, a neonicotinoid insecticide, is widely used for controlling various species of pests on many crops. The potential genotoxic effects of thiacloprid on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and cytokinesis‐block micronucleus (MN) assays. The human PBLs were treated with 75, 150, and 300 μg/mL thiacloprid in the absence and presence of an exogenous metabolic activator (S9 mix). Thiacloprid increased the CAs and SCEs significantly at all concentrations (75, 150, and 300 μg/mL) both in the absence and presence of the S9 mix and induced a significant increase in MN and nucleoplasmic bridge formations at all concentrations for 24 h and at 75 and 150 μg/mL for 48‐h treatment periods in the absence of the S9 mix; and at all concentrations in the presence of the S9 mix when compared with the control and solvent control. Thiacloprid was also found to significantly induce nuclear bud (NBUD) formation at 300 μg/mL for 24 h and at 150 μg/mL for 48‐h treatment times in the absence of the S9 mix and at the two highest concentrations (150 and 300 μg/mL) in the presence of the S9 mix. Thiacloprid significantly decreased the mitotic index, proliferation index, and nuclear division index for all concentrations both in the absence and presence of the S9 mix. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 631–641, 2014.     

In vitro and in vivo human metabolism of the synthetic cannabinoid AB‐CHMINACA

N‐[(1S)‐1‐(aminocarbonyl)‐2‐methylpropyl]‐1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamide (AB‐CHMINACA) is a recently introduced synthetic cannabinoid. At present, no information is available about in vitro or in vivo human metabolism of AB‐CHMINACA. Therefore, biomonitoring studies to screen AB‐CHMINACA consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated the in vitro metabolism of AB‐CHMINACA using human liver microsomes (HLMs). Formation of AB‐CHMINACA metabolites was monitored using liquid chromatography coupled to time‐of‐flight mass spectrometry. Twenty‐six metabolites of AB‐CHMINACA were detected including seven mono‐hydroxylated and six di‐hydroxylated metabolites and a metabolite resulting from N‐dealkylation of AB‐CHMINACA, all produced by cytochrome P450 (CYP) enzymes. Two carboxylated metabolites, likely produced by amidase enzymes, and five glucuronidated metabolites were also formed. Five mono‐hydroxylated and one carboxylated metabolite were likely the major metabolites detected. The involvement of individual CYPs in the formation of AB‐CHMINACA metabolites was tested using a panel of seven human recombinant CYPs (rCYPs). All the hydroxylated AB‐CHMINACA metabolites produced by HLMs were also produced by the rCYPs tested, among which rCYP3A4 was the most active enzyme. Most of the in vitro metabolites of AB‐CHMINACA were also present in urine obtained from an AB‐CHMINACA user, therefore showing the reliability of the results obtained using the in vitro metabolism experiments conducted to predict AB‐CHMINACA in vivo metabolism. The AB‐CHMINACA metabolites to target in biomonitoring studies using urine samples are now reliably identified and can be used for routine analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

In vitro evaluation of the toxicity and underlying molecular mechanisms of Janus Fe3O4‐TiO2 nanoparticles in human liver cells

Recent studies show that Janus Fe₃O₄‐TiO₂ nanoparticles (NPs) have potential applications as a multifunctional agent of magnetic resonance imaging (MRI) and photodynamic therapy (PDT) for the diagnosis and therapy of cancer. However, little work has been done on their biological effects. To evaluate the toxicity and underlying molecular mechanisms of Janus Fe₃O₄‐TiO₂ nanoparticles, an in vitro study using a human liver cell line HL‐7702 cells was conducted. For comparison, the Janus Fe₃O₄‐TiO₂ NPs parent material TiO₂ NPs was also evaluated. Results showed that both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs decreased cell viability and ATP levels when applied in treatment, but increased malonaldehyde (MDA) and reactive oxygen species (ROS) generation. Mitochondria JC‐1 staining assay showed that mitochondrial membrane permeability injury occurred in both NPs treated cells. Cell viability analysis showed that TiO₂ NPs induced slightly higher cytotoxicity than Fe₃O₄‐TiO₂ NPs in HL7702 cells. Western blotting indicated that both TiO₂ NPs and Fe₃O₄‐TiO₂ NPs could induce apoptosis, inflammation, and carcinogenesis related signal protein alterations. Comparatively, Fe₃O₄‐TiO₂ NPs induced higher signal protein expressions than TiO₂ NPs under a high treatment dose. However, under a low dose (6.25 μg/cm²), neither NPs had any significant toxicity on HL7702 cells. In addition, our results suggest both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs could induce oxidative stress and have a potential carcinogenetic effect in vitro. Further studies are needed to elaborate the detailed mechanisms of toxicity induced by a high dose of Fe₃O₄‐TiO₂ NPs.     

Genotoxic effects of a particular mixture of acetamiprid and α‐cypermethrin on chromosome aberration,sister chromatid exchange,and micronucleus formation in human peripheral blood lymphocytes

The genotoxic effects of a particular mixture of acetamiprid (Acm, neonicotinoid insecticide) and α‐cypermethrin (α‐cyp, pyrethroid insecticide) on human peripheral lymphocytes were examined in vitro by chromosomal aberrations (CAs), sister chromatid exchange (SCE), and micronucleus (MN) tests. The human peripheral lymphocytes were treated with 12.5 + 2.5, 15 + 5, 17.5 + 7.5, and 20 + 10 μg/mL of Acm+α‐cyp, respectively, for 24 and 48 h. The mixture of Acm+α‐cyp induced the CAs and SCEs at all concentrations and treatment times when compared with both the control and solvent control and these increases were concentration‐dependent in both treatment times. MN formation was significantly induced at 12.5 + 2.5, 15 + 5, 17.5 + 7.5, μg/mL of Acm+α‐cyp when compared with both controls although these increases were not concentration‐dependent. Binuclear cells could not be detected sufficiently in the highest concentration of the mixture (20 + 10 μg/mL) for both the 24‐ and 48‐h treatment times. Mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) significantly decreased because of the cytotoxic and cytostatic effects of the mixture, at all concentrations for two treatment periods. Significant decreases in MI and PI were concentration dependent at both treatment times. The decrease in NDI was also concentration‐dependent at 48‐h treatment period. In general, Acm+α‐cyp inhibited nuclear division more than positive control, mitomycin C (MMC) and showed a higher cytostatic effect than MMC. Furthermore, in this article, the results of combined effects of Acm+α‐cyp were compared with the results of single effects of Acm or α‐cyp (Kocaman and Topaktas, 2007 , 2009 , respectively). In conclusion, the particular mixture of Acm+α‐cyp synergistically induced the genotoxicity/cytotoxicity in human peripheral blood lymphocytes. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Revision of the affinity constant for perchlorate binding to the sodium‐iodide symporter based on in vitro and human in vivo data

A series of previously published physiologically based pharmacokinetic (PBPK) models describe the effect of perchlorate on iodide uptake by the thyroid, with the mechanism being competitive inhibition of iodide transport by the sodium‐iodide symporter (NIS). Hence a key parameter of these models is the affinity of perchlorate for the NIS, characterized as the Michaelis–Menten kinetic constant, K_m. However, when model predictions were compared to published results of a human study measuring radio‐iodide uptake (RAIU) inhibition after controlled perchlorate exposures, it was found to only fit the lowest exposure level and underpredicted RAIU inhibition at higher levels. Published in vitro data, in which perchlorate‐induced inhibition of iodide uptake via the NIS was measured, were re‐analyzed. K_m for binding of perchlorate to the NIS originally derived from these data, 1.5 μm , had been obtained using Lineweaver–Burk plots, which allow for linear regression but invert the signal–noise of the data. Re‐fitting these data by non‐linear regression of the non‐inverted data yielded a 60% lower value for the K_m, 0.59 μm . Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg^?1 day^?1. Thus, this lower K_m value both fits the in vitro NIS kinetics and provides better predictions of human in vivo RAIU data. This change in K_m increases the predicted sensitivity of humans to perchlorate over twofold for low‐level exposures. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Collecting e‐cigarette aerosols for in vitro applications: A survey of the biomedical literature and opportunities to increase the value of submerged cell culture‐based assessments

Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced‐risk alternatives to the continued use of combustible tobacco products. Because of the widespread uptake of ENDS—in particular, e‐cigarettes—the biological effects, including the toxic potential, of their aerosols are under investigation. Preclinically, collection of such aerosols is a prerequisite for testing in submerged cell culture‐based in vitro assays; however, despite the growth in this research area, there is no apparent standardized collection method for this application. To this end, through an Institute for in vitro Sciences, Inc. workshop initiative, we surveyed the biomedical literature catalogued in PubMed® to map the types of methods hitherto used and reported publicly. From the 47 relevant publications retrieved, we identified seven distinct collection methods. Bubble‐through (with aqueous solvents) and Cambridge filter pad (CFP) (with polar solvents) collection were the most frequently cited methods (57% and 18%, respectively), while the five others (CFP + bubble‐through; condensation; cotton filters; settle‐upon; settle‐upon + dry) were cited less often (2–10%). Critically, the collected aerosol fractions were generally found to be only minimally characterized chemically, if at all. Furthermore, there was large heterogeneity among other experimental parameters (e.g., vaping regimen). Consequently, we recommend that more comprehensive research be conducted to identify the method(s) that produce the fraction(s) most representative of the native aerosol. We also endorse standardization of the aerosol generation process. These should be regarded as opportunities for increasing the value of in vitro assessments in relation to predicting effects on human health.     

Discovery of potential Toxoplasma gondii CDPK1 inhibitors with new scaffolds based on the combination of QSAR and scaffold‐hopping method with in vitro validation

To discover drugs for toxoplasmosis with less side‐effects and less probability to get drug resistance is eagerly appealed for pregnant women, infant or immunocompromised patients. In this work, using TgCDPK1 as drug target, we design a method to discover new inhibitors for CDPK1 as potential drug lead for toxoplasmosis with novel scaffolds based on the combination of 2D/3D‐QSAR and scaffold‐hopping methods. All the binding sites of the potential inhibitors were checked by docking method, and only the ones that docked to the most conserved sites of TgCDPK1, which make them have less probability to get drug resistance, were remained. As a result, 10 potential inhibitors within two new scaffolds were discovered for TgCDPK1 with experimentally verified inhibitory activities in micromole level. The discovery of these inhibitors may contribute to the drug development for toxoplasmosis. Besides, the pipeline which is composed in this work as the combination of QSAR and scaffold‐hopping is simple, easy to repeat for researchers without need of in‐depth knowledge of pharmacology to get inhibitors with novel scaffolds, which will accelerate the procedure of drug discovery and contribute to the drug repurposing study.     

Clinical assessment of the lag‐time and tmax of pellets with controlled release of glucose: in vitro/in vivo comparison using 13C–breath test

Maintaining a stable glycaemia in diabetes mellitus type 1 requires flexible insulin administration and carbohydrate intake to affected individuals. In real life, there might be some situations limiting the insulin–sugar balance control, e.g. night sleep or prolonged sporting activities. Glucose pellets with a pre‐determined time lag between the pellet administration and glucose release were developed to mimic a ‘snack eaten in advance’. In this article, a ¹³C–glucose breath test was introduced to translate laboratory dissolution testing to clinical confirmation of the glucose release pattern using 5% δ abundance to differentiate the appearance of in ¹³C exhaled breath. An independent two‐sample t‐test (p  = 0.20) confirmed an average clinical lag time of 300 min and an in vitro time of 338 min to be identical at a level of significance of α = 0.05. Moreover, using the same statistical method, the clinical t_max (564 min) and the in vitro t₅₀ (594 min) were also considered identical (p  = 0.34). It was concluded that dissolution testing is a relevant method to determine the time lags of dosage forms with controlled release of glucose and that the ¹³C–glucose breath test is a suitable clinical tool for lag time verification in clinical studies.     

Characterization of preclinical in vitro and in vivo ADME properties and prediction of human PK using a physiologically based pharmacokinetic model for YQA‐14, a new dopamine D3 receptor antagonist candidate for treatment of drug addiction

YQA‐14 is a novel and selective dopamine D3 receptor antagonist, with potential for the treatment of drug addiction. However, earlier compounds in its structural class tend to have poor oral bioavailability. The objectives of this study were to characterize the preclinical absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK) of YQA‐14, then to simulate the clinical PK of YQA‐14 using a physiologically based pharmacokinetics (PBPK) model to assess the likelihood of developing YQA‐14 as a clinical candidate. For human PK prediction, PBPK models were first built in preclinical species, rats and dogs, for validation purposes. The model was then modified by input of human in vitro ADME data obtained from in vitro studies. The study data showed that YQA‐14 is a basic lipophilic compound, with rapid absorption (T_max ~ 1 h) in both rats and dogs. Liver microsomal clearances and in vivo clearances were moderate in rats and dogs consistent with the moderate bioavailability observed in both species. The PBPK models built for rats and dogs simulated the observed PK data well in both species. The PBPK model refined with human data predicted that YQA‐14 would have a clearance of 8.0 ml/min/kg, a volume distribution of 1.7 l/kg and a bioavailability of 16.9%. These acceptable PK properties make YQA‐14 an improved candidate for further research and development as a potential dopamine D3R antagonism for the treatment of drug addiction in the clinic. Copyright © 2014 John Wiley & Sons, Ltd.     

Concluding the trilogy: The interaction of 2,2′‐dihydroxy‐benzophenones and their carbonyl N‐analogues with human glutathione transferase M1‐1 face to face with the P1‐1 and A1‐1 isoenzymes involved in MDR

A series of 2,2′‐dihydroxybenzophenones and their carbonyl N‐analogues were studied as potential inhibitors against human glutathione transferase M1‐1 (hGSTM1‐1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1‐1 within a range of 0‐42% (25 μM). The IC₅₀ values for the two stronger ones, 16 and 13 , were 53.5 ± 5.6 μΜ and 28.5 ± 2.5 μΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1‐1 and hGSTA1‐1 involved in MDR. All but one bind more strongly to A1‐1, than M1‐1 and P1‐1, the latter being a poor binder. An order of potency A1‐1 > > M1‐1 >  P1‐1 meritted 13 , 14 and 16 as the most potent inhibitors with hGSTM1‐1. Enzyme kinetics with hGSTM1‐1 (K_m(CDNB) 213 ± 10 μΜ and K_m(GSH) 303 ± 11 μΜ) revealed a competitive modality for 16 (K_i(16) = 22.3 ± 1.1 μΜ) and a mixed one for 13 versus CDNB (K_i(13) = 33.3 ± 1.6 μM for the free enzyme and K_i(13)′ = 17.7 ± 1.7 μM for the enzyme‐CDNB complex). 5‐ or 5′‐Bromo‐ or phenyl‐substituted (but not in combination) inhibitors, having a H‐bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1‐1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.     

An assessment of the impact of multi‐route co‐exposures on human variability in toxicokinetics: A case study with binary and quaternary mixtures of volatile drinking water contaminants

This study aimed to assess the impact of multi‐route co‐exposures to chemicals on interindividual variability in toxicokinetics. Probabilistic physiologically based pharmacokinetic multi‐route interaction models were developed for adults and four younger subpopulations. Drinking water‐mediated multi‐route exposures were simulated for benzene alone or in co‐exposure with toluene, ethylbenzene and m‐xylene, for trichloroethylene or vinyl chloride (VC), alone and in mixture. These simulations were performed for “low” and “high” exposure scenarios, involving respectively the US EPA's short‐term drinking water health advisories, and 10 times these advisory values. Distributions of relevant internal dose metrics for benzene, trichloroethylene and VC were obtained using Monte Carlo simulations. Intergroup variability indexes (VI) were computed for the “low” (VI_L) and “high” (VI_H) exposure scenarios, as the ratio between the 95th percentile in each subpopulation over the median in adults. Thus, for benzene, parent compound's area under the curve‐based VI_L for single exposures vs. co‐exposures correspondingly varied between 1.7 (teenagers) and 2.8 (infants) vs. 1.9 and 3.1 respectively. VI_H varied between 2.5 and 3.5 vs. 2.9 and 4.1. Inversely, VI_L and VI_H for the amount of benzene metabolized via CYP2E1 pathway decreased in co‐exposure compared to single exposure. For VC and trichloroethylene, similar results were obtained for the “high” exposure, but “low” co‐exposures did not impact the toxicokinetics of individual substances. In conclusion, multi‐route co‐exposures can have an impact on the toxicokinetics of individual substances, but to an extent, that does not seem to challenge the default values attributed to the factors deemed at reflecting interindividual or child/adult differences in toxicokinetics.