Moderate Toxicity of Potential Boron-containing Therapeutic, Dipotassium-trioxohydroxytetrafl uorotriborate - K2 (B3 O3 F4 OH) in Rats and Mice

Abstract Biological activity of boron-containing compounds (BCCs) has been well-known. Growing interest and numerous applications for BCCs have been reported. Boron and boron-containing acids show low acute toxicity in mammals but data on halogenated boroxine (HB) - dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH) acute toxicity have not been reported before. This compound, characterized as a potential therapeutic for skin changes, exhibits no observable genotoxicity in doses lower that 0.1 mg/ml in vitro and 55 mg/kg in vivo. It has also been confirmed as an antitumour agent both in vitro and in vivo as well as an inhibitor of enzymes involved in antioxidant mechanisms. The aim of this study was to assess the acute toxicity of HB and to determine the maximum tolerated dose as well as a dose free of any signs of toxicity in different test organisms. Acute toxicity of HB was tested in Sprague-Dawley and Wistar rats and BALB/c mice after single parenteral application of different doses. We determined doses free of any sign of toxicity and LD50 after single dose administration. LD50 of HB ranges from 63 to 75 mg/kg in different test models, meaning that HB shows moderate toxicity

Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.

BACKGROUND: Local antibiotic application has been widely used in orthopedic surgery. The dose-related toxicity of antibiotics towards periosteal tissues and resulting effects on osteogenic expression are yet to be studied. METHODS: Periosteal cells harvested from the medial tibia of New Zealand White rabbits were used. A seeding density of 5 × 103 cells/cm2 was determined to be optimal for testing in the pilot study; the cells were cultured in xCELLigence 96-well plates. Microfluidic impedance analyzers were used to monitor cellular proliferation in microfluidic culture systems with exposure to three different concentrations (10 µg/mL, 100 µg/mL, and 1000 µg/mL) of cefazolin, ciprofloxacin, and vancomycin, respectively. The correlation of cell index at day 7 with optical density values from WST-1 assays using conventional cultures was evaluated by calculating the Pearson's coefficient. RNA analysis was performed to investigate the expression of osteogenic markers in the cultured cells, including core-binding factor alpha 1 (Cbfa1), osteopontin (OPN), and osteopontin promoter (OPNp), relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control. RESULTS: A significant dose-related inhibition of cell index was found for all the 3 antibiotics, whereas the WST-1 assays showed a significant dose-related inhibition of cellular proliferation only at a high dose of cefazolin (1000 µg/mL) and medium-to-high dose of ciprofloxacin (100 µg/mL and 1000 µg/mL). Pearson's coefficient analysis indicated a high correlation between the cell index and optical density values of WST-1 assays only for medium and high doses of ciprofloxacin (100 µg/mL and 1000 µg/mL); a moderate correlation was seen for cefazolin, and a low dose of ciprofloxacin (10 µg/mL). RNA analysis confirmed significant dose-related inhibition of cfba1, OPN, and OPNp expression by all three antibiotics. CONCLUSION: With optimal seeding amounts, rabbit periosteal cells can be dynamically monitored in the xCELLigence microfluidic system. Dose-related inhibition of cellular proliferation and osteogenic expression was found after exposure to cefazolin and ciprofloxacin. By providing real-time detection and exhibiting comparable correlation, microfluidic impedance-based analyzer is a feasible alternative to the conventional WST-1 assays.

Nonanimal Models for Acute Toxicity Evaluations: Applying Data-Driven Profiling and Read-Across.

BACKGROUND: Low-cost, high-throughput in vitro bioassays have potential as alternatives to animal models for toxicity testing. However, incorporating in vitro bioassays into chemical toxicity evaluations such as read-across requires significant data curation and analysis based on knowledge of relevant toxicity mechanisms, lowering the enthusiasm of using the massive amount of unstructured public data. OBJECTIVE: We aimed to develop a computational method to automatically extract useful bioassay data from a public repository (i.e., PubChem) and assess its ability to predict animal toxicity using a novel bioprofile-based read-across approach. METHODS: A training database containing 7,385 compounds with diverse rat acute oral toxicity data was searched against PubChem to establish in vitro bioprofiles. Using a novel subspace clustering algorithm, bioassay groups that may inform on relevant toxicity mechanisms underlying acute oral toxicity were identified. These bioassays groups were used to predict animal acute oral toxicity using read-across through a cross-validation process. Finally, an external test set of over 600 new compounds was used to validate the resulting model predictivity. RESULTS: Several bioassay clusters showed high predictivity for acute oral toxicity (positive prediction rates range from 62-100%) through cross-validation. After incorporating individual clusters into an ensemble model, chemical toxicants in the external test set were evaluated for putative acute toxicity (positive prediction rate equal to 76%). Additionally, chemical fragment -in vitro-in vivo relationships were identified to illustrate new animal toxicity mechanisms. CONCLUSIONS: The in vitro bioassay data-driven profiling strategy developed in this study meets the urgent needs of computational toxicology in the current big data era and can be extended to develop predictive models for other complex toxicity end points. https://doi.org/10.1289/EHP3614.

Application of Daphnia magna for Nanoecotoxicity Study.

Daphnia magna (D. magna), an aquatic invertebrate, is the most commonly used test organism in ecotoxicological studies on manufactured nanomaterials (MNs). Although standard protocols for undertaking acute and chronic toxicity tests of dissolved chemicals with D. magna have been endorsed by several national and international organizations, comprehensive guidance on aquatic toxicology testing of MNs with D. magna are in their infancy. Here we describe the acute and chronic toxicity methods with D. magna as test organism for nanoecotoxicity study. These methods may provide reference for the next step toward developing prescriptive aquatic toxicity standard tests for MNs with D. magna.

The abdominal skin of female Sprague-Dawley rats is more sensitive than the back skin to drug-induced phototoxicity.

In vivo phototoxicity studies are important to predict drug-induced phototoxicity in humans; however, a standard methodology has not established. To determine differences in sensitivity to drug-induced phototoxicity among various skin sites, we evaluated phototoxic reactions in the back and abdominal skin of female Sprague-Dawley rats orally dosed with phototoxic drugs (pirfenidone, 8-methoxysoraren, doxycycline, and lomefloxacin) or a non-phototoxic drug (gatifloxacin) followed by solar-simulated light irradiation comprising 18J/cm2 ultraviolet A. Tissue reactions were evaluated by macroscopic and microscopic examination and immunohistochemistry for γ-H2AX, and tissue concentrations of pirfenidone, doxycycline, and lomefloxacin were measured by tandem mass spectrometry. In addition, the thicknesses of the skin layers at both sites were measured in drug-naïve rats. The abdominal skin showed more severe reactions to all phototoxic drugs than the back skin, whereas the minimal erythema dose in drug-naïve rats and skin concentrations of each drug were comparable between the sites. Furthermore, histopathological lesions and γ-H2AX-positive cells in the abdominal skin were detected in deeper layers than in the back skin. The stratum corneum and dermis in the abdominal skin were significantly thinner than in the back skin, indicating a difference in the depth of light penetration and potentially contributing to the site differences observed in sensitivity to phototoxicity. Gatifloxacin did not induce any phototoxic reactions at either site. In conclusion, the abdominal skin is more sensitive to drug-induced phototoxicity than the back skin and may represent a preferable site for irradiation in this rat phototoxicity model.

Methods for the Detection of Autophagy in Mammalian Cells.

Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. © 2016 by John Wiley & Sons, Inc.

In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.

A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.

A high throughput passive dosing format for the Fish Embryo Acute Toxicity test.

High throughput testing according to the Fish Embryo Acute Toxicity (FET) test (OECD Testing Guideline 236) is usually conducted in well plates. In the case of hydrophobic test substances, sorptive and evaporative losses often result in declining and poorly controlled exposure conditions. Therefore, our objective was to improve exposure conditions in FET tests by evaluating a passive dosing format using silicone O-rings in standard 24-well polystyrene plates. We exposed zebrafish embryos to a series of phenanthrene concentrations until 120h post fertilization (hpf), and obtained a linear dilution series. We report effect values for both mortality and sublethal morphological effects based on (1) measured exposure concentrations, (2) (lipid normalized) body residues and (3) chemical activity. The LC50 for 120hpf was 310µg/L, CBR50 (critical body residue) was 2.72mmol/kg fresh wt and La50 (lethal chemical activity) was 0.047. All values were within ranges expected for baseline toxicity. Impaired swim bladder inflation was the most pronounced morphological effect and swimming activity was reduced in all exposure concentrations. Further analysis showed that the effect on swimming activity was not attributed to impaired swim bladder inflation, but rather to baseline toxicity. We conclude that silicone O-rings (1) produce a linear dilution series of phenanthrene in the 120hpf FET test, (2) generate and maintain aqueous concentrations for reliable determination of effect concentrations, and allow for obtaining mechanistic toxicity information, and (3) cause no toxicity, demonstrating its potential as an extension of the FET test when testing hydrophobic chemicals.

Evaluation of the ecotoxicity of pollutants with bioluminescent microorganisms.

This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.

Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests.

Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000 mg/kg b.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration-response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained.

Acute nose-only inhalation exposure of rats to di- and triphosgene relative to phosgene.

Groups of young adult Wistar rats were acutely exposed to trichloromethyl chloroformate (diphosgene) and bis(trichloromethyl) carbonate (triphosgene) vapor atmospheres using a directed-flow nose-only mode of exposure. The exposure duration used was 240 min. The median lethal concentration (LC50) of diphosgene and triphosgene was 13.9 and 41.5 mg/m3, respectively. Based on the molar exposure concentrations, the LC50s of phosgene (previously published), diphosgene, and triphosgene were 0.07, 0.07, and 0.14 mmol/m3, respectively. Although the principal toxic mode of action of the volatile diphosgene was similar to phosgene gas, the vapor phase of triphosgene appeared to be different to that of phosgene and diphosgene based on a more persistent occurrence of signs of respiratory distress and a biphasic onset of mortality. While all substances caused mortality within 1 day postexposure, triphosgene induced a second phase of mortality 11?14 days postexposure. The vapor saturation concentration of triphosgene at ambient temperature is ?100 times its LC50. In summary, triphosgene-induced lung injury patterns are different from that of phosgene and diphosgene. More research is needed to close the substantial data gaps of triphosgene.

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH - Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.

Acute airway effects of ozone-initiated d-limonene chemistry: importance of gaseous products.

There are concerns about ozone-initiated chemistry, because the formation of gaseous oxidation products and ultrafine particles may increase complaints, morbidity and mortality. Here we address the question whether the gaseous products or the ultrafine particles from the ozone-initiated chemistry of limonene, a common and abundant indoor pollutant, cause acute airway effects. The effects on the airways by d-limonene, a ca. 16s old ozone/d-limonene mixture, and clean air were evaluated by a mice bioassay, from which sensory irritation of the upper airways, airflow limitation, and pulmonary irritation can be obtained. A denuder was inserted to separate the ultrafine particles from the gaseous products prior to the exposure chamber. Reduction of mean respiratory frequency (>30%) and 230% increase of time of brake were observed without denuder, during 30min exposure, to the ozonolyzed d-limonene mixture, which are indicative of prominent sensory effects. The initial concentrations (ppm) were 40 d-limonene and 4 ozone. The exposure concentrations (ppm) were about 35 d-limonene and 0.05 ozone. Formaldehyde and residual d-limonene, the salient sensory irritants, accounted for up to three-fourth of the sensory irritation. The upper airway effects reversed to baseline upon cessation of exposure. An effect on the conducting airways was also significant, which did not reverse completely upon cessation. Airway effects were absent with the denuder inserted, which did not alter the size distribution of ultrafine particles ( approximately 10mg/m(3)), significantly. The result was statistically indistinguishable from clean dry air. It is concluded that ultrafine particles that are generated from ozone-initiated d-limonene chemistry and denuded are not causative of sensory effects in the airways.

Effects of nickel chloride and oxygen depletion on behaviour and vitality of zebrafish (Danio rerio, Hamilton, 1822) (Pisces, Cypriniformes) embryos and larvae.

We examined acute (2 h exposure of 5-day-old larvae) and subchronic (exposure from fertilization up to an age of 11 days) effects of NiCl(2).6H2O on embryos and larvae of zebrafish (Danio rerio), both alone and in combination with oxygen depletion. The following endpoints were recorded: acute exposure: locomotory activity and survival; subchronic exposure: hatching rate, deformations, locomotory activity (at 5, 8 and 11 days) and mortality. In acute exposures nickel chloride (7.5-15 mg Ni/L) caused decreasing locomotory activity. Oxygen depletion (or=10 mg Ni/L resulted in delayed hatching at an age of 96 h, in decreased locomotory activity at an age of 5 days, and increased mortality at an age of 11 days (LC20=9.5 mg Ni/L). The observed LOEC for locomotory activity (7.5 mg Ni/L) is in the range of environmentally relevant concentrations. Since locomotory activity was already affected by acute exposure, this parameter is recommended to supplement commonly recorded endpoints of toxicity.

Mosquito coil smoke inhalation toxicity. Part I: validation of test approach and acute inhalation toxicity.

Burning mosquito coils indoors to repel mosquitoes is a common practice in many households in tropical countries. The evaluation and assessment of the inhalation toxicity of smoke emitted from mosquito coils appear to be particularly challenging due to the complex nature of this type of exposure atmosphere. The potential health implications of the gases, volatile agents and particulate matter emitted from burning coils or incense have frequently been addressed; however, state-of-the-art inhalation toxicity studies are scarce. The focus of this paper was comparatively to evaluate and assess the appropriateness and practical constraints of the whole-body versus the nose-only mode of exposure for inhalation toxicity studies with burning mosquito coils. With regard to the controlled exposure of laboratory animals to complex smoke atmospheres the nose-only mode of exposure had distinct advantages over the whole-body exposure, which included a rapid attainment of the inhalation chamber steady state, minimization of particle coagulation and uncontrolled adsorption of condensate onto the chamber surfaces. While in whole-body chambers a different kinetic behaviour of volatile and particulate constituents was found which caused inhomogeneous, i.e. artificially enriched atmospheres with volatile components at the expense of aerosols, the nose-only mode of exposure provided maximum exposure intensities without losses of the particulate phase of the exposure atmosphere. Collectively, the results obtained support the conclusion that the dynamic nose-only mode of exposure is experimentally superior to the quasistatic whole-body exposure mode which provides the least control over exposure atmospheres and the outcome highly contingent on selected experimental factors. Acute inhalation toxicity studies in rats suggest that the most critical metrics of exposure are apparently related to (semi)volatile upper respiratory tract sensory irritants, whilst the asphyxic component, carbon monoxide, plays a role only at overtly irritant exposure levels. However, this study was conducted at exposure concentrations much higher than encountered by people in residential settings and the effects observed under these conditions may not be relevant to hazards from exposures at common use levels. Neither an acute 8 h exposure of rats nor the 1 h sensory irritation study in mice and rats provided experimental evidence that irritant particle-related effects had occurred in the lower respiratory tract. In summary, the protocols devised evaluate and assess the acute inhalation toxicity of mosquito coil smoke demonstrating that the nose-only mode of exposure of rats to the smoke of mosquito coils is suitable to assess the toxic potency of different coils. The nose-only mode has clear advantages over the whole-body exposure mode. The inhalation studies conducted show unequivocally that acute toxic effects are difficult to produce with this type of product even under rigorous testing conditions.

Retrospective analysis of acute inhalation toxicity studies: comparison of actual concentrations by filter and cascade impactor analyses.

Determination of acute inhalation toxicity is usually the initial step in the assessment and evaluation of the toxic characteristic of a substance that may be inhaled. Commonly, data from this bioassays may serve as a basis for classification and labeling and may also be used for the derivation of Emergency Response Guidance Levels. The focus of this analysis is on the comparative measurement of actual total mass concentrations in inhalation chambers obtained from independent filter (or alternative) analyses and cascade impactor analyses and whether the similarity/disparity of concentration measurements found by different equipment and sampling strategies could serve as robust criterion for the identification of inconclusive measurements. Potential artifacts leading to erroneous concentrations include anisokinetic sampling errors, obstructions of filters, errors related to the calculation/measurement of the sampled volume of atmospheres, wall losses or evaporation. The outcome of this analysis supports the conclusion that the mass concentrations obtained by the commonly performed cascade impactor analysis provide an important adjunct to the established procedures. In summary, the similarity of mass concentrations obtained independently by cascade impactor and filter analyses, i.e., sampling equipment with different aspiration efficiencies and collection media, improve the judgment whether the results from atmosphere characterization are 'conclusive' or 'inconclusive.'

[A system for predicting the acute toxicity of chemical substances].

A method has been proposed to recognize and predict the range values of acute toxicity (LD50) and for this a system has been developed. The essence of the method is to gradually decrease the predictable toxicity ranges for the test substances in a stepwise manner while advancing within the formed predictive complexes. The transmission of information on the structure of compounds along this complex is determined by the preset logistic scheme along which narrower LD50 ranges are designated. The intervals are automatically set, by optimizing the recognition of the properties of compounds at the stage of formation of models (that recognize the set of signs). The predicting system includes two predicting complexes and contains a total of 30 mathematical models: for heterocyclic and carbocyclic compounds. Logistic schemes for passage of predictable compounds have been developed for each complex in accordance with the hierarchic organization of the elements that are constituents of the complex. The prediction significance is 70-90%.

Static and dynamic acute cytotoxicity assays on microfluidic devices.

Static and dynamic acute toxicity assays of cells were performed on microfluidic devices where materials were hydraulically transported. Static assays were performed by incubating cells with an agent in a microchip reservoir and optically interrogating the cells after hydrodynamic focusing at a cross intersection. Dynamic assays were performed on a microchip with a 25-cm-long spiral channel where the cells were mixed with an agent and optically monitored 0.1, 12, and 22 cm from the point of mixing. The incubation time was determined by the time needed for cells to transit from the mixing location to the point of detection. Cell viability was determined using the ratio of fluorescence signals from membrane permeant (calcein) and membrane impermeant (propidium iodide) stains. The model system used in this study was the viability of Jurkat cells in the presence of the agent Triton X-100). An average LC50 value of 138 microM for Triton X-100 was obtained for an incubation period of 7-12 min using the static assay. LC50 values obtained with the dynamic assay for 25- and 47-s incubation times were 290 and 250 microM Triton X-100, respectively. Higher LC50 values for the dynamic assay were expected due to the shorter incubation times.