Applied In Vitro Toxicology

The Vitrocell^® Ames 48 whole-aerosol exposure module offers a high-throughput platform for air-agar-interface exposures that mimic human exposure conditions for assessing potential mutagenic activity of a test article. The aim of this study was to characterize the Vitrocell Ames 48 exposure module for assessment of aerosolized tobacco products utilizing multiple dosimetry techniques and nicotine deposition as determined by chemical analysis.

Characterization was performed using the Vitrocell VC10^® smoking machine and three nicotine containing tobacco product test articles: Kentucky Reference cigarettes (3R4F), a commercially available electrically heated tobacco product (eHTP), and an Electronic Nicotine Delivery System (ENDS). The following dosimetry tools were used: Aerosol photometers (using area under curve values, ∑AUC), free glycerol deposition in phosphate-buffered saline (PBS), fluorescence of anhydrous dimethyl sulfoxide (DMSO)-captured smoke particulate matter, and analytical determination of nicotine in PBS and DMSO. Characterization of 3R4F Kentucky Reference cigarettes was examined over a diluting airflow range 8 to 0.5 L/min; eHTP and ENDS were examined over a diluting airflow range of 4 to 0 L/min (undiluted).

Results from the dosimetry techniques showed that whole smoke from 3R4F cigarettes and whole aerosol from eHTP and ENDS demonstrated reproducible and consistent delivery, with results being consistent between experiments and within each airflow.

These results show that this exposure module is fit for purpose using these three tobacco product types.

Coculture models have been extensively used for assessing the toxicity of fibers and particles. However, once cell lines are mixed in the same tissue culture well, it is difficult to evaluate differential cell toxicity without using specific cell markers, which might not be compatible with assays requiring living cells such as a particle-induced oxidative stress assay by flow cytometry.

Human alveolar epithelial cells (A549) were specifically labeled with cell proliferation dye—eFluor™ 670—before being mixed with phorbol ester-differentiated Tohoku Hospital Pediatrics-1 cells (used as macrophages). The coculture model allowed the toxicity of crystalline silica DQ-12 and DQ-12-PVNO (particles were coated with polyvinylpyridine-N-oxide, a molecule used to quench particle surface reactivity) to be assessed. Particle-induced oxidative stress was evaluated by flow cytometry using a 2′,7′-dichlorodihydrofluorescein diacetate probe (H₂DCFDA).

A549 cell treatment with a noncytotoxic concentration of an eFluor 670 probe allowed labeled and unlabeled cells to be differentiated using flow cytometry. Cellular oxidative stress induced by phorbol ester or DQ-12 detected by H₂DCFDA was not affected by eFluor 670 probe cell treatment.

This study showed that specific labeling of live cells before coculture setup allows assays such as oxidative stress assessment by flow cytometry to be conducted on different live cell types from the same coculture models without the use of cell-type-specific markers. Such assays would be of great value in any kind of multiple cell type model in which the effects of chemicals on a given cell population is sought.

Allelopathic effects have a wide range of action, at the genetic and biochemical level, favoring the germination and survival of plants and, often, helping as a phytosanitary strategy in reducing infections that generate loss in the agro-industrial sector. However, in addition to pesticides, metabolites of natural origin have been identified as an important tool in combating phytopathogenic microorganisms and, due to their symbiotic activities in the environment, growth promoters of several plant species. This study aims to assess the antimicrobial activity against Burkholderia cepacia, allelopathic, and phytotoxic potential of the (+)− usnic acid (UA).

The experiments were conducted in laboratory conditions using the compound at different concentrations and the test organism were Solanum lycopersicum.

The compound at 12.5 μg/mL was able to reduce >90% of the evaluated bacterial population and any significant toxic effects were observed on the germination.

In addition, at 25 μg/mL, the UA did not interfere in the fresh and dry weight of the tomato, corroborating the potential of this compound as a biocontroller, since it does not cause a pronounced phytotoxic effect, selectively assessing a pathogenic microorganism.